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教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次 专业



授课内容 氨基酸代谢 授课方式及学时 讲授 8 学时





目的要求

目 1. 了解蛋白质的营养作用及消化吸收与腐败作用。

的 2. 了解氨基酸的主要生理功能。掌握体内氨基酸的代谢概况。重点

要 掌握氨基酸的脱氨基作用及α－酮酸的代谢 。

求 3. 重点掌握氨的来源与去路，氨的转运，尿素合成的部位及主要过

、 程。

重 4 掌握熟悉氨基酸的脱羧基作用，重点掌握一碳单位的概念、种类、

点 功能以及一碳单位与四氢叶酸的关系。 掌握甲硫氨基循环及作用，

与 掌握酪氨酸的代

难 5 了解支链氨基酸代谢。

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参 1 Instant notes of Biochemistry 2nd edition

考 2 生物化学 第 6 版

书 3 LEHNINGER 生物化学原理

目









1 A small fraction of oxidative energy in humans comes from the catabolism

of amino acids. Amino acids are derived from the normal breakdown

(recycling) of cellular proteins, degradation of ingested proteins, or

breakdown of body proteins in lieu of other fuel sources during starvation

or in untreated diabetes mellitus. Ingested proteins are degraded in the

stomach and small intestine by proteases. Most proteases are initially

synthesized as inactive zymogens, which are activated in the stomach or

intestine by proteolytic removal of parts of their polypeptide chains.

An early step in the catabolism of amino acids is the separation of the

amino group from the carbon skeleton. In most cases, the amino group is

transferred to α-ketoglutarate to form glutamate. This type of reaction

is called a transamination and requires the coenzyme pyridoxal phosphate.

Glutamate is transported to liver mitochondria, where an amino group is

liberated as ammonia (NH4+ ) by the enzyme glutamate dehydrogenase. Ammonia

formed in other tissues is transported to liver mitochondria as the amide

nitrogen of glutamine or as the amino group of alanine. Most of the alanine

is generated in muscle and transported in the blood to the liver. After

deamination the resulting pyruvate is converted to glucose, which is

transported back to muscle as part of the glucose-alanine cycle.



2 Ammonia is highly toxic to animal tissues. Ammonotelic animals (bony

fishes, tadpoles) excrete amino nitrogen from their gills as ammonia.

Ureotelic animals (adult terrestrial amphibians and all mammals) excrete

amino nitrogen as urea, formed in the liver by the urea cycle. Arginine

is the immediate precursor of urea. Arginase hydrolyzes arginine to yield

urea and ornithine, and arginine is resynthesized in the urea cycle.

Ornithine is converted to citrulline at the expense of carbamoyl phosphate,

and an amino group is transferred to citrulline from aspartate, re-forming

arginine. Ornithine is regenerated in each turn of the cycle. Several of

the intermediates and byproducts of the urea cycle are also intermediates

in the citric acid cycle, and the two cycles are thus interconnected. The

activity of the urea cycle is regulated at the levels of enzyme synthesis

and allosteric regulation of the enzyme that forms carbamoyl phosphate.

Uricotelic animals (birds and reptiles) excrete amino nitrogen in

semisolid form as uric acid, a derivative of purine. The mode of nitrogen

excretion is determined by habitat. The formation of the nontoxic urea

and of solid uric acid has a high ATP cost. Genetic defects in enzymes

of the urea cycle can be compensated for by dietary regulation.



3 After removal of amino groups by transamination to α-ketoglutarate,

the carbon skeletons of amino acids undergo oxidation to compounds that

can enter the citric acid cycle for oxidation to CO2 and H2O. In these

pathways, the cofactors tetrahydrofolate and S-adenosylmethionine

facilitate one-carbon transfer reactions, and the cofactor

tetrahydrobiopterin facilitates the oxidation of phenylalanine catalyzed

by phenylalanine hydroxylase. There are five intermediates through which

carbon skeletons of amino acids enter the citric acid cycle: (1)

acetyl-CoA, (2) α-ketoglutarate, (3) succinylCoA, (4) fumarate, and (5)

oxaloacetate. The amino acids producing acetyl-CoA are divided into two

groups. Alanine, cysteine, glycine, tryptophan, and serine yield

acetyl-CoA via pyruvate; leucine, lysine, phenylalanine, tyrosine, and

tryptophan yield acetyl-CoA via acetoacetyl-CoA. Isoleucine, leucine,

and tryptophan also form acetyl-CoA directly. Proline, histidine,

arginine, glutamine, and glutamate enter the citric acid cycle via

α-ketoglutarate; threonine, methionine, isoleucine, and valine enter

via succinyl-CoA; four carbon atoms of phenylalanine and tyrosine enter

via fumarate; and asparagine and aspartate enter via oxaloacetate. The

branched-chain amino acids (leucine, isoleucine, and valine), unlike the

other amino acids, are degraded in extrahepatic tissues. A number of

serious human diseases can be traced to genetic defects in specific

enzymes in the pathways of amino acid catabolism.Some amino acids can be

converted to ketone bodies; some can be converted to glucose.



4 Mammals (e.g., humans and the albino rat) can synthesize 10 of the 20

amino acids of proteins. The remainder, which are required in the diet

(essential amino acids), can be synthesized by plants and bacteria. Among

the nonessential amino acids, glutamate is formed by reductive amination

of α-ketoglutarate and is the precursor of glutamine, proline, and

arginine. Alanine and aspartate (and thus asparagine) are formed from

pyruvate and oxaloacetate, respectively, by transamination. The carbon

chain of serine is derived from 3-phosphoglycerate. Serine is a precursor

of glycine; the β-carbon atom of serine is transferred to

tetrahydrofolate. Cysteine is formed from methionine and serine by a

series of reactions in which S-adenosylmethionine and cystathionine are

intermediates. The aromatic amino acids (phenylalanine, tyrosine, and

tryptophan) are formed via a pathway in which the intermediate chorismate

occupies a key branch point. Phosphoribosyl pyrophosphate is a precursor

of tryptophan and histidine, both essential amino acids. The biosynthetic

pathway to histidine is interconnected with the purine synthetic pathway.

Tyrosine can also be formed by hydroxylation of phenylalanine, an

essential amino acid. The pathways for biosynthesis of the other essential

amino acids in bacteria and plants are complex. The amino acid

biosynthetic pathways are subject to allosteric end-product inhibition;

the regulatory enzyme is usually the first in the sequence. The regulation

of these synthetic pathways is coordinated.



5 Many other important biomolecules are derived from amino acids. Glycine

is a precursor of porphyrins; porphyrins, in turn, are degraded to form

bile pigments. Glycine and arginine give rise to creatine and

phosphocreatine. Glutathione, a tripeptide, is an important cellular

reducing agent. DAmino acids are synthesized from L-amino acids in

bacteria in racemization reactions requiring pyridoxal phosphate. The

PLP-dependent decarboxylation of certain amino acids yields some

important biological amines, including neurotransmitters. The aromatic

amino acids are precursors of a number of plant substances.

6 The amino acid and nucleotide biosynthetic pathways make repeated use

of the biological cofactors pyridoxal phosphate, tetrahydrofolate, and

S-adenosylmethionine. Pyridoxal phosphate is required for transamination

reactions involving glutamate and for a number of other amino acid

transformations. One-carbon transfers are carried out using

S-adenosylmethionine (at the -CH3 oxidation level) and tetrahydrofolate

(usually at the -CHO and -CH2OH oxidation levels). Enzymes called

glutamine amidotransferases are used in reactions that incorporate

nitrogen derived from glutamine.







教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 蛋白质 授课方式及学时 讲授 8 学时





目的要求

目 1. 了解蛋白质的重要生理功能。

的 2. 掌握蛋白质的化学组成；氮的平均含量及换算；蛋白质的基本结

要 构单位――氨基酸。了解氨基酸的分类及重要氨基酸的分子结构。

求 3. 重点掌握肽键、肽键平面、多肽链、蛋白质的一级结构、二级结

、 构、三级结构、四级结构的概念，二级结构的种类及特点。

重 4. 掌握蛋白质分子结构与功能的关系。

点 5. 重点掌握蛋白质的两性电离及等电点、高分子性质、变性、沉淀

与 等概念及其与医学的关系。

难 6. 了解蛋白质的分类及氨基酸序列测定。

点 教学内容

1、蛋白质的分子组成

2、蛋白质的分子结构

3、蛋白质的结构与功能的关系

4、蛋白质的理化性质及其分离纯化





课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计

参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理







1 The 20 amino acids commonly found as hydrolysis products of proteins

contain an α-carboxyl group, an α-amino group, and a distinctive R group

substituted on the α-carbon atom. The α-carbon atom of the amino acids

(except glycine) is asymmetric, and thus amino acids can exist in at least

two stereoisomeric forms. Only the L stereoisomers, which are related to

the absolute configuration of L-glyceraldehyde, are found in proteins.



2 The amino acids are classified on the basis of the polarity of their

R groups. The nonpolar, aliphatic class includes alanine, glycine,

isoleucine, leucine, proline, and valine. Phenylalanine, tryptophan, and

tyrosine have aromatic side chains and are also relatively hydrophobic.

The polar, uncharged class includes asparagine, cysteine, glutamine,

methionine, serine, and threonine. The negatively charged (acidic) amino

acids are aspartate and glutamate; the positively charge (basic) ones are

arginine, histidine, and lysine. There are also a large ndmber of

nonstandard amino acids that occur in some proteins (as a result of the

modification of standard amino acids) or as free metabolites in cells.



3 Monoamino monocarboxylic amino acids are diprotic acids (+H3NCH(R)COOH)

at low pH. As the pH is raised to about 6, near the isoelectric point,

the proton is lost from the carboxyl group to form the dipolar or

zwitterionic species +H3NCH(R)COO-, which is electrically neutral. Further

increase in pH causes loss of the second proton, to yield the ionic species

H2NCH(R)COO-. Amino acids with ionizable R groups may exist in additional

ionic species, depending on the pH and the pKa of the R group. Thus amino

acids vary in their acid-base properties. Amino acids form colored

derivatives with ninhydrin. Other colored or fluorescent derivatives are

formed in reactions of the α-amino group of amino acids with

fluorescamine, dansyl chloride, dabsyl chloride, and

1-fluoro-2,4-dinitrobenzene. Complex mixtures of amino acids can be

separated and identified by ionexchange chromatography or HPLC.



4 Amino acids can be joined covalently through peptide bonds to form

peptides, which can also be formed by incomplete hydrolysis of

polypeptides. The acid-base behavior and chemical reactions of a peptide

are functions of its amino-terminal amino group, its carboxyl-terminal

carboxyl group, and its R groups. Peptides can be hydrolyzed to yield free

amino acids. Some peptides occur free in cells and tissues and have

specific biological functions. These include some hormones and

antibiotics, as well as other peptides with powerful biological activity.



5 Cells generally contain thousands of different proteins, each with a

different function or biological activity. These functions include

enzymatic catalysis, molecular transport, nutrition, cell or organismal

motility, structural roles, organismal defense, regulation, and many

others. Proteins consist of very long polypeptide chains having from 100

to over 2,000 amino acid residues joined by peptide linkages. Some

proteins have several polypeptide chains, which are then referred to as

subunits. Simple proteins yield only amino acids on hydrolysis;

conjugated proteins contain in addition some other component, such as a

metal ion or organic prosthetic group.



6 Every protein has a unique three-dimensional structure that reflects

its function, a structure stabilized by multiple weak interactions.

Hydrophobic interactions provide the major contribution to stabilizing

the globular form of most soluble proteins; hydrogen bonds and ionic

interactions are optimized in the specific structure that is

thermodynamically most stable.



7 There are four generally recognized levels of protein structure. Primary

structure refers to the amino acid sequence and the location of disulfide

bonds. Secondary structure refers to the spatial relationship of adjacent

amino acids. Tertiary structure is the three-dimensional conformation of

an entire polypeptide chain. Quaternary structure involves the spatial

relationship of multiple polypeptide chains (e.g., enzyme subunits) that

are tightly associated.



8 The nature of the bonds in the polypeptide chain places constraints on

structure. The peptide bond is characterized by a partial double-bond

character that keeps the entire amide group in a rigid planar

configuration. The N-Cα and Cα-C bonds can rotate with bond angles φ and

ψ, respectively. Secondary structure can be defmed completely by these

two bond angles.



9 There are two general classes of proteins: fibrous and globular. Fibrous

proteins, which serve mainly structural roles, have simple repeating

structures and provided excellent models for the early studies of protein

structure. Two major types of secondary structure were predicted by model

building based on information obtained from fibrous proteins: the α helix

and the β conformation. Both are characterized by optimal hydrogen

bonding between amide nitrogens and carbonyl oxygens in the peptide

backbone. The stability of these structures within a protein is influenced

by their amino acid content and by the relative placement of amino acids

in the sequence. Another nonrepeating type of secondary structure common

in proteins is the β bend.



10 In fibrous proteins such as keratin and collagen, a single type of

secondary structure predominates. The polypeptide chains are

supertwisted into ropes and then combined in larger bundles to provide

strength. The structure of elastin permits stretching.



11 Globular proteins have more complicated tertiary structures, often

containing several types of secondary structure in the same polypeptide

chain. The first globular protein structure to be determined, using x-ray

diffraction methods, was that of myoglobin. This structure confirmed that

a predicted secondary structure (α helix) occurs in proteins; that

hydrophobic amino acids are located in the protein interior; and that

globular proteins are compact. Subsequent research on protein structure

has reinforced these conclusions while demonstrating that different

proteins often differ in tertiary structure.



12 The three-dimensional structure of proteins can be destroyed by

treatments that disrupt weak interactions, a process called denaturation.

Denaturation destroys protein function, demonstrating a relationship

between structure and function. Some denatured proteins (e.g.,

ribonuclease) can renature spontaneously to give active protein, showing

that the tertiary structure of a protein is determined by its amino acid

sequence.



13 The folding of globular proteins is believed to begin with local

formation of regions of secondary structure, followed by interactions of

these regions and adjustments to reach the final tertiary structure.

Sometimes regions of a polypeptide chain, called domains, fold up

separately and can have separate functions. The final structure and the

steps taken to reach it are influenced by the need to bury hydrophobic

amino acid side chains in the protein interior away from water, the

tendency of a polypeptide chain to twist in a right-handed sense, and the

need to maximize hydrogen bonds and ionic interactions. These constraints

give rise to structural patterns such as the β-α-β fold and twisted

β pleated sheets. Even at the level of tertiary structure, some common

patterns are found in proteins that have no known functional relationship.



14 Quaternary structure refers to the interaction between the subunits

of oligomeric proteins or large protein assemblies. The best-studied

oligomeric protein is hemoglobin. The four subunits of hemoglobin exhibit

cooperative interactions on oxygen binding. Binding of oxygen to one

subunit facilitates oxygen binding to the next, giving rise to a sigmoid

binding curve. These effects are mediated by subunit-subunit interactions

and subunit conformational changes. Very large protein structures

consisting of many copies of one or a few dif ferent proteins are referred

to as supramolecular complexes. These are found in cellular skeletal

structures, muscle and other types of cellular "engines," and virus coats.



15 Proteins are purified by taking advantage of properties in which they

differ, such as size, shape, binding affmities, charge, etc. Purification

also requires a method for quantifying or assaying a particular protein

in the presence of others. Proteins can be both separated and visualized

by electrophoretic methods. Antibodies that specifically bind a certain

protein can be used to detect and locate that protein in a solution, a

gel, or even in the interior of a cell.



16 All proteins are made from the same set of 20 amino acids. Their

differences in function result from differences in the composition and

sequence of their amino acids. The amino acid sequences of polypeptide

chains can be established by fragmenting them into smaller pieces using

several specific reagents, and determining the amino acid sequence of each

fragment by the Edman degradation procedure. The sequencing of suitably

sized peptide fragments has been automated. The peptide fragments are then

placed in the correct order by finding sequence overlaps between fragments

generated by different methods. Protein sequences can also be deduced from

the nucleotide sequence of the corresponding gene in the DNA. The amino

acid sequence can be compared with the thousands of known sequences, often

revealing insights into the structure, function, cellular location, and

evolution of the protein.



17 Homologous proteins from different species show sequence homology:

certain positions in the polypeptide chains contain the same amino acids,

regardless of the species. In other positions the amino acids may differ.

The invariant residues are evidently essential to the function of the

protein. The degree of similarity between amino acid sequences of

homologous proteins from different species correlates with the

evolutionary relationship of the species.





教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 蛋白质的生物合成—翻译 授课方式及学时 讲授 8 学时

目的要求

目 1. 重点掌握蛋白质生物合成的概况：原料、三类 RNA 在蛋白质生

的 物合成中的作用。遗传密码的概念及特点。

要 2. 掌握蛋白质合成的基本过程：氨基酸的活化与搬运，起始、延

求 长及终止，核蛋白体循环。

、 3. 了解翻译后的加工修饰。

重 4. 了解蛋白质合成的干扰与抑制。

点

与

难

点









课

1.多媒体教学。

堂



设

2.重点内容进行归纳。

计









参 1 Instant notes of Biochemistry 2nd edition

考 2 生物化学 第 6 版

书 3 LEHNINGER 生物化学原理

目





1 Proteins are synthesized with a particular amino acid sequence through

the translation of information encoded in messenger RNA by an RNAprotein

complex called a ribosome. Amino acids are specified by informational

units in the mRNA called codons. Translation requires adapter molecules,

the transfer RNAs, which recognize codons and insert amino acids into

their appropriate sequential positions in the polypeptide.



2 The codons for the amino acids consist of specific nucleotide triplets.

The base sequences of the codons were deduced from experiments using

synthetic mRNAs of known composition and sequence. The genetic code is

degenerate: it has multiple code words for nearly all the amino acids.

The third position in each codon is much less specific than the first and

second and is said to wobble. The standard genetic code words are probably

universal in all species, although some minor deviations exist in

mitochondria and a few single-celled organisms. The initiating amino acid,

N-formylmethionine in bacteria, is coded by AUG. Recognition of a

particular AUG as the initiation codon requires a purine-rich initiating

signal (the Shine-Dalgarno sequence) on the 5' side of the AUG. The

triplets UAA, UAG, and UGA do not code for amino acids but are signals

for chain termination. In some viruses two different proteins may be coded

by the same nucleotide sequence but translated with dif ferent reading

frames.



3 Protein synthesis occurs on the ribosomes. Bacteria have 70S ribosomes,

with a large (50S) subunit and a small (30S) subunit. Ribosomes of

eukaryotes are significantly larger and contain more proteins than do

bacterial ribosomes.



4 In stage 1 of protein synthesis, amino acids are activated by specific

aminoacyl-tRNA synthetases in the cytosol. These enzymes catalyze the

formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP

and PPi. The fidelity of protein synthesis depends to a large extent on

the accuracy of this reaction, and some of these enzymes carry out

proofreading steps at separate active sites. Transfer RNAs have 73 to 93

nucleotide units, several of which have modified bases. They have an amino

acid arm with the terminal sequence CCA(3') to which an amino acid is

esterified, an anticodon arm, a T?C arm, and a DHU arm; some tRNAs have

a fifth or extra arm. The anticodon nucleotide triplet of tRNA is

responsible for the specificity of interaction between the aminoacyltRNA

and the complementary codon on the mRNA. The growth of polypeptide chains

on ribosomes begins with the amino-terminal amino acid and proceeds by

successive additions of new residues to the carboxyl-terminal end.



5 In bacteria, the initiating aminoacyl-tRNA in all proteins is

N-formylmethionyl-tRNAfMet. Initiation of protein synthesis (stage 2)

involves formation of a complex between the 30S ribosomal subunit, mRNA,

GTP, fMet-tRNAfMet, two initiation factors, and the 50S subunit; GTP is

hydrolyzed to GDP and Pi. In the subsequent elongation steps (stage 3),

GTP and three elongation factors are required for binding the incoming

aminoacyl-tRNA to the aminoacyl site on the ribosome. In the first

peptidyl transfer reaction, the fMet residue is transferred to the amino

group of the incoming aminoacyl-tRNA. Movement of the ribosome along the

mRNA then translocates the dipeptidyl-tRNA from the aminoacyl site to the

peptidyl site, a process requiring hydrolysis of GTP. After many such

elongation cycles, synthesis of the polypeptide chain is terminated

(stage 4) with the aid of release factors. A polysome consists of an mRNA

molecule to which are attached several or many ribosomes, each

independently reading the mRNA and forming a polypeptide. At least four

high-energy phosphate bonds are required to generate each peptide bond,

an energy investment required to guarantee fidelity of translation. In

stage 5 of protein synthesis, polypeptides undergo folding into their

active, three-dimensional forms. Many proteins also are further processed

by posttranslational modification reactions.



6 After synthesis, many proteins are directed to particular locations in

the cell. One targeting mechanism involves peptide signal sequences

generally found at the amino terminus of newly synthesized proteins. In

eukaryotes, one class of these signal sequences is recognized and bound

by a large protein-RNA complex called the signal recognition particle

(SRP). The SRP binds the signal sequence as soon as it appears on the

ribosome and transfers the entire ribosome and incomplete polypeptide to

the endoplasmic reticulum. Polypeptides with these signal sequences are

moved into the lumen of the endoplasmic reticulum as they are synthesized;

there they may be modified and moved to the Golgi complex, and then sorted

and sent to lysosomes, the plasma membrane, or secretory vesicles. Other

known targeting signals include carbohydrates (mannose-6-phosphate

targets proteins to lysosomes) and three-dimensional structural features

of the proteins called signal patches. Some proteins are imported into

the cell by receptor-mediated endocytosis. These receptors are also used

by some toxins and viruses to gain entry into cells.



7 Proteins are eventually degraded by specialized proteolytic systems

present in all cells. Defective proteins and those slated for rapid

turnover are generally degraded by an ATP-dependent proteolytic system.

In eukaryotes, proteins to be broken down by this system are first tagged

by linking them to a highly conserved protein called ubiquitin.







教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 核苷酸代谢 授课方式及学时 讲授 8 学时

目的要求

1. 了解核苷酸的生理功能与消化吸收。

目 2. 重点掌握嘌呤核苷酸、嘧啶核苷酸的从头合成的原料及基本途

的 径。了解补救合成途径及调节。

要 3. 掌握核苷酸的抗代谢物及临床应用。

求 4. 掌握核苷酸的分解代谢，重点掌握核苷酸分解代谢的终产物。

、

重

点

与

难

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理









1 The purine ring system is built up in a step-bystep fashion on

5-phosphoribosylamine. The amino acids glutamine, glycine, and aspartate

furnish all the nitrogen atoms of purines.



2 Two ring-closure steps ensue to form the purine nucleus. Pyrimidines

are synthesized from carbamoyl phosphate and aspartate.

Ribose-5-phosphate is then attached to yield the pyrimidine

ribonucleotides.



3 Purine and pyrimidine biosynthetic pathways are regulated by feedback

inhibition. Nucleoside monophosphates are converted to their

triphosphates by enzymatic phosphorylation reactions. Ribonucleotides

are converted to deoxyribonucleotides by the action of ribonucleotide

reductase, an enzyme with novel mechanistic and regulatory

characteristics.



4 The thymine nucleotides are derived from the deoxyribonucleotides dCDP

and dUMP. Uric acid and urea are the end products of purine and pyrimidine

degradation. Free purines can be salvaged and rebuilt into nucleotides

by a separate pathway.



5 Genetic deficiencies in certain salvage enzymes cause serious genetic

diseases such as Lesch-Nyhan syndrome and severe immunodeficiency disease.

Another genetic deficiency results in the accumulation of uric acid

crystals in the joints, causing gout.



6 The enzymes of the nucleotide biosynthetic pathways are targets for an

array of chemotherapeutic agents used to treat cancer and other diseases.







教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 核 酸 授课方式及学时 讲授 8 学时





目的：

目 1. 掌握核酸的分类、细胞内分布及生物学功能。

的 2、了解核酸的元素组成，平均磷含量及换算。

要 3、掌握核苷酸、核苷、碱基的基本概念及其结构。

求 4、掌握核酸的紫外吸收特性，核酸变性、复性及杂交等概念。

、

重 重点：

点 1. 掌握核酸的分类、细胞内分布及生物学功能。

与 2、了解核酸的元素组成，平均磷含量及换算。

难 3、掌握核苷酸、核苷、碱基的基本概念及其结构。

点 4、掌握核酸的紫外吸收特性，核酸变性、复性及杂交等概念

难点：

DNA 的三级结构――核小体。三类 RNA――rRNA、mRNA、tRNA

的结构特点及功能

课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理









1 Nucleotides serve a diverse set of important functions in cells. As

subunits of nucleic acids they carry genetic information. They are also

the primary carriers of chemical energy in cells, structural components

of many enzyme cofactors, and cellular second messengers.



2 A nucleotide consists of a nitrogenous base (purine or pyrimidine), a

pentose sugar, and one or more phosphate groups. Nucleic acids are

polymers of nucleotides, linked together by phosphodiester bridges

between the 5' hydroxyl of one pentose and the 3' hydroxyl of the next.

There are two types of nucleic acid: RNA and DNA. The nucleotides in RNA

contain ribose, and the common pyrimidine bases are uracil and cytosine.

In DNA, the nucleotides contain 2'-deoxyribose, and the pyrimidine bases

are generally thymine and cytosine. The primary purines are adenosine and

guanine in both RNA and DNA.



3 Many lines of evidence show that DNA bears genetic information. In

particular, the AveryMacLeod-McCarty experiment showed that DNA isolated

from one strain of a bacterium can enter and transform the cells of another

strain, endowing it with some of the inheritable characteristics of the

donor. The Hershey-Chase experiment showed that the DNA of a bacterial

virus, but not its protein coat, carries the genetic message for

replication of the virus in the host cell.



4 From x-ray diffraction studies of DNA fibers and the base equivalences

in DNA discovered by Chargaff (A = T and G ≡ C), Watson and Crick

postulated that native DNA consists of two antiparallel chains in a

right-handed double-helical arrangement. Complementary base pairs, A=T

and G≡C, are formed by hydrogen bonding within the helix, and the

hydrophilic sugar-phosphate backbones are located on the outside. The

base pairs are stacked perpendicular to the long axis, 0.34 nm apart; there

are about 10 base pairs in each complete turn of the double helix.



5 DNA can exist in several structural forms. Two variations from the

Watson-Crick B-form DNA, the A and Z forms, have been characterized in

DNA crystal structures. The A-form helix is shorter and of greater

diameter than a B-form helix with the same sequence. The Z form is a

lefthanded helix. Some sequence-dependent structural variations cause

bends in the DNA. DNA strands with self-complementary inverted repeats

can form hairpin or cruciform structures. Polypyrimidine tracts arranged

in mirror repeats can take up a triple-helical structure called H-DNA.



6 Messenger RNA is the vehicle by which genetic information is transferred

to ribosomes for protein synthesis. Transfer RNA and ribosomal RNA are

also involved in protein synthesis. RNA can be structurally complex, with

single RNA strands often folded into hairpins, double-stranded regions,

and complex loops.



7 Native DNA undergoes reversible unwinding and separation (melting) of

strands on heating or at extremes of pH. Because G≡C base pairs are more

stable than A=T pairs, the melting point of DNAs rich in G≡C pairs is

higher than that of DNAs rich in A=T pairs. Denatured singlestranded DNAs

from two species can form a hybrid duplex, the degree of hybridization

depending on the extent of sequence homology. Hybridization is the basis

for important techniques used to study and isolate specific genes and

RNAs.



8 DNA is a relatively stable polymer. Very slow, spontaneous reactions

such as deamination of certain bases, hydrolysis of base-sugar

N-glycosidic bonds, formation of pyrimidine dimers (radiation damage),

and oxidative damage are important because of the very low tolerance of

cells for changes in genetic material. DNA sequences can be determined

and DNA polymers synthesized using simple protocols involving chemical

and enzymatic methods.



9 ATP is the central carrier of chemical energy in cells, probably

reflecting the requirement for binding energy in catalysis. The presence

of adenosine in the structure of a variety of enzyme cofactors may also

be related to binding energy requirements. Cyclic AMP is a common second

messenger produced in response to hormones and other chemical signals.

It is formed from ATP in a reaction catalyzed by adenylate cyclase.

教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 酶 授课方式及学时 讲授 8 学时





目的要求

目 1. 掌握酶的基本概念：化学本质，酶催化作用的特点。

的 2. 重点掌握酶的结构与活性：酶的化学组成――酶蛋白、辅助因

要 子、全酶、酶的活性中心和必需基团，酶原与酶原的激活，同工

求 酶以及酶活性的调节。

、 3. 掌握酶的作用原理：活化能、中间产物学说的概念。

重 4. 重点掌握酶促反应动力学的基本内容：温度、PH、酶浓度、底

点 物浓度、激活剂及抑制剂对酶催化反应的影响。米氏方程，米氏

与 常数的意义。

难 5. 掌握酶活性测定的基本原则，酶活性单位的概念。

点 6. 了解酶与医学的关系，酶与疾病的关系，酶在医学上的应用。

7. 了解酶的命名与分类。

教学内容

1、酶的分子结构与功能

2、酶促反应的特点与机制

3、酶促反应动力学

4、酶的调节

5、酶与医学的关系





课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理

1 Virtually every biochemical reaction is catalyzed by enzymes. With the

exception of a few catalytic RNAs, all known enzymes are proteins. Enzymes

are extraordinarily effective catalysts, commonly producing reaction

rate enhancements of 107 to 1014. To be active, some enzymes require a

chemical cofactor, which can be loosely or tightly bound. Each enzyme is

classified according to the specific reaction it catalyzes.



2 Enzyme-catalyzed reactions are characterized by the formation of a

complex between substrate and enzyme (an ES complex). The binding occurs

in a pocket on the enzyme called the active site. The function of enzymes

and other catalysts is to lower the activation energy for the reaction

and thereby enhance the reaction rate. The equilibrium of a reaction is

unaffected by the enzyme.



3 The energy used for enzymatic rate enhancements is derived from weak

interactions (hydrogen bonds and van der Waals, hydrophobic, and ionic

interactions) between the substrate and enzyme. The enzyme active site

is structured so that many of these weak interactions occur only in the

reaction transition state, thus stabilizing the transition state. The

energy available from the numerous weak interactions between enzyme and

substrate (the binding energy) is substantial and can generally account

for observed rate enhancements. The need for multiple interactions is one

reason for the large size of enzymes. Binding energy can be used to lower

substrate entropy, to strain the substrate, or to cause a conformational

change in the enzyme (induced fit). This same binding energy accounts for

the exquisite specificity exhibited by enzymes for their substrates.

Other catalytic mechanisms include general acid-base catalysis and

covalent catalysis. Details of the reaction mechanisms have been worked

out for many enzymes.



4 Kinetics is an important method for the study of enzyme mechanisms. Most

enzymes have some common kinetic properties. As the concentration of the

substrate is increased, the catalytic activity of a fixed concentration

of an enzyme will increase in a hyperbolic fashion to approach a

characteristic maximum rate Vmax, at which essentially all the enzyme is

in the form of the ES complex. The substrate concentration giving one-half

Vmax is the Michaelis-Menten constant Km, which is characteristic for each

enzyme acting on a given substrate. The Michaelis-Menten equation

V0=Vmax[S]/(Km+[S]) relates the initial velocity of an enzymatic reaction

to the substrate concentration and Vmax through the constant Km. Both Km and

Vmax can be measured; they have different meanings for different enzymes.

The limiting rate of an enzyme-catalyzed reaction at saturation is

described by the constant kcat, also called the turnover number. The ratio

Kcat/Km provides a good measure of catalytic efficiency. The

Michaelis-Menten equation is also applicable to bisubstrate reactions,

which occur by either ternary complex or double-displacement (ping-pong)

pathways. Each enzyme has an optimum pH, as well as a characteristic

specificity for the substrates on which it acts.



5 Enzymes can be inactivated by irreversible modification of a functional

group essential for catalytic activity. They can also be reversibly

inhibited, competitively or noncompetitively. Competitive inhibitors

compete reversibly with the substrate for binding to the active site, but

they are not transformed by the enzyme. Noncompetitive inhibitors bind

to some other site on the free enzyme or to the ES complex.



6 Some enzymes regulate the rate of metabolic pathways in cells. In

feedback inhibition, the end product of a pathway inhibits the first

enzyme of that pathway. The activity of some regulatory enzymes, called

allosteric enzymes, is adjusted by reversible, noncovalent binding of a

specific modulator to a regulatory or allosteric site. Such modulators

may be inhibitory or stimulatory and may be either the substrate itself

or some other metabolite. The kinetic behavior of allosteric enzymes

reflects cooperative interactions among the enzyme subunits. Other

regulatory enzymes are modulated by covalent modification of a specific

functional group necessary for activity, or by proteolytic cleavage of

a zymogen.



教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 糖代谢 授课方式及学时 讲授 8 学时



目的要求

1. 了解糖的重要生理功能。

目 2. 了解糖的消化与吸收。

的 3. 掌握糖的无氧分解基本反应过程、关键酶、ATP 的生成、生理意

要 义及调节。

求 4、 重点掌握糖的有氧氧化基本反应过程、关键酶、ATP 的生成、三

、 羧酸循环的特点及生理意义。

重 5、了解 磷酸戊糖途径基本反应过程。掌握关键酶及生理意义。

点 6、掌握糖原的合成与分解基本反应过程、生理意义及调节。

与 7、掌握糖异生作用概念，基本反应过程。掌握关键酶、了解调节及

难 生理意义。

点 8、掌握血糖的代谢来源与去路、激素对血糖浓度的调节。

9、了解高血糖与低血糖。掌握糖尿病时糖代谢的障碍。

课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理







1 Glycolysis is a universal metabolic pathway for the catabolism of

glucose to pyruvate accompanied by the formation of ATP. The process is

catalyzed by ten cytosolic enzymes, and all of the intermediates are

phosphorylated compounds. In the preparatory phase of glycolysis, ATP is

invested to convert glucose to the phosphorylated intermediate

fructose1,6-bisphosphate, then the carbon-carbon bond between C-3 and C-4

is broken to yield two molecules of triose phosphate. In the payoff phase

of glycolysis, each of the two molecules of glyceraldehyde-3-phosphate

derived from glucose undergoes oxidation at C-1; the energy of this

oxidation reaction is conserved in the formation of NADH and an acyl

phosphate bond in 1,3-bisphosphoglycerate. This compound has a high

phosphate group transfer potential, and in a substrate-level

phosphorylation catalyzed by phosphoglycerate kinase the phosphate group

is transferred to ADP, forming ATP and 3-phosphoglycerate. Rearrangement

of the atoms in 3-phosphoglycerate with the loss of H2O gives rise to

phosphoenolpyruvate, another compound with high phosphate group transfer

potential. Phosphoenolpyruvate donates a phosphate group to ADP to form

ATP in the second substratelevel phosphorylation; the other product of

this reaction is pyruvate, the end product of the payoff phase of

glycolysis. The overall equation for glycolysis is



1. Glucose + 2NAD+ + 2ADP + 2Pi 2 pyruvate + 2NADH + 2H+ + 2ATP

+ 2H2O



There is a net gain of two ATP.

2 The NADH formed in glycolysis must be recycled to regenerate NAD+, which

is required as electron acceptor in the first step of the payoff phase

of glycolysis. Under aerobic conditions, electrons pass from NADH to O2

through a chain of electron carriers in the process of mitochondrial

respiration. Under anaerobic conditions, many organisms regenerate NAD+

by transferring electrons from NADH to pyruvate, forming lactate. This

process occurs in vertebrate muscle during intense muscular activity,

when energy demand outstrips the ability to deliver O2 to the muscles.

Other organisms, such as yeast, regenerate NAD+ by reducing pyruvate to

ethanol and CO2. In these anaerobic processes, called fermentations, no

net oxidation or reduction of the carbons of glucose occurs. A variety

of alcohols and organic acids are produced commercially by exploiting the

ability of microorganisms to ferment glucose to these products.



3 Pyruvate, the end product of glycolysis, undergoes dehydrogenation and

decarboxylation by the pyruvate dehydrogenase complex, which contains

three sequentially acting enzymes and requires five coenzymes, to yield

acetyl-CoA and CO2. The acetyl-CoA enters the citric acid cycle, which

occurs in the mitochondria of eukaryotes and in the cytosol of prokaryotes.

Citrate synthase catalyzes the condensation of acetyl-CoA with

oxaloacetate to form citrate. Aconitase catalyzes the reversible

formation of isocitrate from citrate; isocitrate is then oxidized to

α-ketoglutarate by isocitrate dehydrogenase in a reaction that also

yields CO2. The α-ketoglutarate undergoes another dehydrogenation and

decarboxylation to succinyl-CoA and CO2. Succinyl-CoA reacts with ADP (or

GDP) and Pi to form free succinate and ATP (or GTP), in a substrate-level

phosphorylation. The succinate is then oxidized to fumarate by succinate

dehydrogenase, an FAD-linked enzyme that is part of the inner membrane

of the mitochondrion (or of the plasma membrane in bacteria). Fumarate

is reversibly hydrated by fumarase to L-malate, which is oxidized by

NAD-linked L-malate dehydrogenase to regenerate a molecule of

oxaloacetate. The latter can now combine with another molecule of

acetylCoA and start another turn of the cycle.



4 Isotopic tracer experiments with carbon-labeled fuel molecules or

intermediates have established that the citric acid cycle is the major

pathway of carbohydrate oxidation in aerobic cells. The pyruvate

dehydrogenase complex of vertebrates is inhibited by the allosteric

effectors NADH, ATP, and acetyl-CoA. The enzyme complex is also inhibited

by reversible phosphorylation catalyzed by a protein kinase and

phosphatase that are part of the complex. The overall rate of the cycle

is controlled by the rate of conversion of pyruvate to acetyl-CoA and by

the flux through three enzymes of the cycle: citrate synthase, isocitrate

dehydrogenase, and α-ketoglutarate dehydrogenase. These fluxes are

largely determined by the concentrations of substrates and products; the

end products ATP and NADH are inhibitory.



5 Citric acid cycle intermediates are also used as precursors in

biosynthesis of amino acids and other biomolecules. The cycle

intermediates are then replenished by anaplerotic reactions catalyzed by

pyruvate carboxylase, PEP carboxykinase, PEP carboxylase, or malic enzyme.

In the germinating seeds of some plants, and in certain microorganisms

that can live on acetate as sole carbon source for the synthesis of

carbohydrate, a variation of the citric acid cycle, the glyoxylate cycle,

comes into play. The process involves two additional enzymes: isocitrate

lyase and malate synthase, located within glyoxysomes. This cycle makes

possible the net formation of succinate, oxaloacetate, and other cycle

intermediates from acetyl-CoA. Oxaloacetate thus formed can be used to

synthesize glucose via gluconeogenesis. Vertebrates lack the glyoxylate

cycle and cannot synthesize glucose from acetate. In organisms with both

the citric acid cycle and the glyoxylate cycle, the partitioning of

isocitrate between the two pathways is controlled at the level of

isocitrate dehydrogenase. This enzyme is subject to regulation by

reversible phosphorylation.



6 Gluconeogenesis is the formation of carbohydrate from noncarbohydrate

precursors, the most important of which are pyruvate, lactate, and alanine.

In vertebrates, gluconeogenesis in the liver and kidney provides glucose

for use by the brain, muscle, and erythrocytes. Like all biosynthetic

pathways, gluconeogenesis proceeds by an enzymatic route that differs

from the corresponding catabolic pathway, is independently regulated, and

requires ATP. The biosynthetic pathway from pyruvate to glucose occurs

in all organisms. It employs seven of the glycolytic enzymes, which

function reversibly. Three irreversible steps in the glycolytic pathway

cannot be used in gluconeogenesis in the cell, and these are bypassed by

reactions catalyzed by nonglycolytic enzymes: conversion of pyruvate into

phosphoenolpyruvate via oxaloacetate, involving several enzymes and two

high-energy phosphate groups; dephosphorylation of

fructose-1,6-bisphosphate by fructose-1,6-bisphosphatase; and

dephosphorylation of glucose-6-phosphate by glucose-6phosphatase. The

path from pyruvate to phosphoenolpyruvate varies somewhat depending upon

whether lactate or pyruvate itself serves as the gluconeogenic precursor.

Formation of one molecule of glucose from pyruvate requires four molecules

of ATP and two of GTP. Three carbon atoms of each of the citric acid cycle

intermediates and some or all carbons of many of the amino acids are

convertible into glucose. Gluconeogenesis in the liver is regulated at

two major points: (1) the carboxylation of pyruvate by pyruvate

carboxylase, which is stimulated by the allosteric effector acetyl-CoA,

and (2) the dephosphorylation of fructose-1,6-bisphosphate by

fructose-1,6-bisphosphatase, which is inhibited by

fructose-2,6-bisphosphate and AMP and stimulated by citrate.

Fructose-2,6-bisphosphate also stimulates the glycolytic enzyme

phosphofructokinase-1 and is crucial to the balance between

gluconeogenesis and glycolysis. The levels of fructose-2,6-bisphosphate

are hormonally regulated in animals. Reciprocal regulation of

gluconeogenesis and glycolysis prevents futile cycling with its

accompanying loss of ATP energy.



7 Glucose has catabolic fates other than glycolysis. The pentose phosphate

pathway results in oxidation and decarboxylation at the C-1 position of

glucose, producing NADPH and pentose phosphates; NADPH provides reducing

power for biosynthetic reactions, and pentose phosphates are essential

components of nucleotides and nucleic acids. Other oxidative pathways

transform glucose into glucuronic acid and ascorbic acid (vitamin C).

Glucuronidation converts certain nonpolar toxins into polar derivatives

that can be excreted. Humans cannot synthesize ascorbic acid; the lack

of this vitamin in the human diet leads to the disease scurvy.



8 Glycogen and starch, polymeric storage forms of glucose, enter

glycolysis in a two-step process that begins with phosphorolytic cleavage

of a glucose residue from an end of the polymer, forming

glucose-1-phosphate. This is catalyzed by glycogen (or starch)

phosphorylase. Phosphoglucomutase then converts glucose-1-phosphate to

glucose-6-phosphate, the first intermediate in glycolysis. Ingested

disaccharides are converted into monosaccharides in the animal intestine

by specific hydrolytic enzymes on the outer surface of intestinal

epithelial cells; the monosaccharides are then taken up and transported

to the liver or other tissues.



9 Glycogen phosphorylase of vertebrate muscle is activated by

phosphorylation at the Ser14 residue of each subunit, catalyzed by

phosphorylase b kinase, which is itself activated by a cascade of

regulatory events triggered by the hormone epinephrine. Inactivation of

glycogen phosphorylase results from the action of a specific phosphatase

that removes the phosphate groups from the Ser14 residues; this enzyme,

too, is under hormonal regulation. The glycogen phosphorylase of liver

is also regulated by phosphorylation and dephosphorylation, but the

details of its regulation differ from those of the muscle enzyme,

reflecting the difierent roles of muscle and liver in the metabolism of

glucose. Liver serves as a buffer against changes in blood glucose

concentrations; it releases glucose from stored glycogen when the hormone

glucagon signals that blood glucose is too low. The dephosphorylation of

the Ser14 residues in the liver enzyme is stimulated when glucose binds

to an allosteric site on the phosphorylase. When intracellular glucose

rises, signaling that there is suff`icient glucose in the blood, the

glycogen phosphorylase is dephosphorylated and thus inactivated, slowing

the mobilization of free glucose from liver glycogen.



10 Glycogen synthesis also proceeds via a pathway different from its

breakdown. It requires conversion of glucose-1-phosphate into

UDP-glucose, a sugar nucleotide. Sugar phosphates are activated and

earmarked for a particular synthetic path by ester linkage of a nucleoside

diphosphate to the anomeric carbon of the sugar. Glycogen synthase adds

glucose units from UDP-glucose to the nonreducing end of the growing

glycogen chain, forming (α1->4) links. A branching enzyme,

glycosyl-(α1->6)transferase, is necessary to add (α1->6) branch points.

The initiation of glycogen synthesis requires a primer protein called

glycogenin. The synthesis and breakdown of glycogen are reciprocally

regulated by hormone-dependent phosphorylation of glycogen synthase

(inactivating it) and of glycogen phosphorylase (activating it).



11 In a metabolically active cell at steady state, intermediates are

formed and consumed at equal rates. Of paramount importance to a cell is

the maintenance of a high steady-state concentration of ATP. When some

perturbation alters the rate of formation or consumption of an

intermediate or product such as ATP, compensating changes in the

activities of the relevant enzymes bring the system back into the steady

state. These changes in enzyme activity are achieved by allosteric

regulation or covalent modification (such as phosphorylation) of the

enzymes, often triggered by hormonal signals.



12 In multistep processes such as glycolysis, certain of the

enzyme-catalyzed reactions are essentially at equilibrium in the steady

state; the rates of these reactions rise and fall with substrate

concentration, and they are said to be substratelimited. Other reactions

are out of equilibrium; their rates are too slow to produce instant

equilibration of substrate and product, and these reactions are said to

be enzyme-limited. The enzymelimited reactions in a multistep process are

often highly exergonic and therefore practically irreversible, and the

enzymes that catalyze these reactions are commonly the points at which

flux through the pathway is regulated. In the glycolytic pathway from

glycogen to pyruvate, the regulated steps include those catalyzed by

glycogen phosphorylase, hexokinase, phosphofructokinase-1 (PFK-1), and

pyruvate kinase; all are exergonic, enzyme-limited reactions.



13 Hexokinase is inhibited by high concentrations of its product,

glucose-6-phosphate; thus, when the product of the reaction accumulates,

its rate of production is lowered. Pyruvate kinase is likewise

allosterically inhibited by one of its products, ATP.

14 Cellular respiration occurs in three stages: (1) the oxidative

formation of acetyl-CoA from pyruvate, fatty acids, and some amino acids,

(2) the degradation of acetyl residues by the citric acid cycle to yield

CO2 and electrons, and (3) the transfer of electrons to molecular oxygen,

coupled to the phosphorylation of ADP to ATP. The oxidative catabolism



教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 生物氧化 授课方式及学时 讲授 8 学时





目的要求

目 1. 了解生物氧化的概念、特点及有关酶类。

的 2. 掌握呼吸链的组成及线粒体内的两条重要呼吸链——NADH 氧

要 化呼吸链和 FADH 氧化呼吸链。掌握线粒体外 NADH 的氧化。

求 3. 重点掌握氧化磷酸化作用的概念、偶联部位及影响因素。

、 4. 掌握 ATP 与能量的转换和利用。

重

点

与

难

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理

1 Chemiosmotic theory provides the intellectual framework for

understanding many biological energy transductions, including the

processes of oxidative phosphorylation in mitochondria and

photophosphorylation in chloroplasts. The mechanism of energy coupling

is similar in both cases. The conservation of free energy involves the

passage of electrons through a chain of membrane-bound

oxidation-reduction (redox) carriers and the concomitant pumping of

protons across the membrane, producing an electrochemical gradient, the

protonmotive force. This force drives the synthesis of ATP by

membrane-bound enzyme complexes through which protons flow back across

the membrane, down their electrochemical gradient. Protonmotive force

also drives other energy-requiring processes of cells.



2 In mitochondria, H atoms removed from substrates by the action of

NAD-linked dehydrogenases donate their electrons to the respiratory

(electron transfer) chain, which transfers them to molecular O2, reducing

it to H2O. Shuttle systems convey reducing equivalents from cytosolic NADH

to mitochondrial NADH. Reducing equivalents from all NAD-linked

dehydrogenations are transferred to mitochondrial NADH dehydrogenase

(Complex I), which contains FMN as its prosthetic group. They are then

passed via a series of Fe-S centers to ubiquinone, which transfers the

electrons to cytochrome b, the first carrier in Complex III. In this

complex, electrons pass through two b-type cytochromes and cytochrome cl

before reaching an Fe-S center. The Fe-S center passes electrons, one at

a time, through cytochrome c and into Complex IV, cytochrome oxidase. This

copper-containing enzyme, which also contains cytochromes a and a3,

accumulates electrons, then passes them to O2, reducing it to H2O.



3 There are alternative paths of entry of electrons into this chain of

carriers. Succinate, for example, is oxidized by succinate dehydrogenase

(Complex II), which contains a flavoprotein (with FAD) that passes

electrons through several Fe-S centers and into the chain at the level

of ubiquinone. Electrons derived from the oxidation of fatty acids pass

into ubiquinone via the electron-transferring flavoprotein (ETFP).



4 The flow of electrons through Complexes I, III, and IV results in the

pumping of protons across the mitochondrial inner membrane, making the

matrix alkaline relative to the extramitochondrial space. This proton

gradient provides the energy (proton-motive force) for ATP synthesis from

ADP and Pi by an inner-membrane protein complex, ATP synthase, also called

F0F1 ATPase. The details of this ATP-synthesizing mechanism are still under

investigation. Bacteria carry out oxidative phosphorylation by

essentially the same mechanism, using electron carriers and an ATP

synthase in the plasma membrane. Oxidative phosphorylation produces most

of the ATP required by aerobic cells; it is regulated by cellular energy

demands. In brown fat tissue, which is specialized for the production of

metabolic heat, electron transfer is uncoupled from ATP synthesis; the

energy of fatty acid oxidation is therefore dissipated as heat.



5 Photophosphorylation in the chloroplasts of green plants and in

cyanobacteria also involves electron flow through a series of

membrane-bound carriers. In the light reactions of plants, the absorption

of a photon excites chlorophyll molecules and other (accessory) pigments

that funnel the energy into reaction centers in the thylakoid membranes

of chloroplasts. At the reaction centers, photoexcitation results in a

charge separation that produces one chemical species that is a good

electron donor (reducing agent) and another that is a good electron

acceptor. In chloroplasts there are two different photoreaction centers,

which function

together. Photosystem I passes electrons from its excited reaction center,

P700, through a series of carriers to ferredoxin, which then reduces NADP+

to NADPH. The reaction center, P680, of photosystem II passes electrons

to plastoquinone, reducing it to the quinol form. The electrons lost from

P680 are replaced by electrons abstracted from H2O (hydrogen donors other

than H2O are used in other organisms). This light-driven splitting of H2O

is catalyzed by a Mn-containing protein complex; O2 is produced. Reduced

plastoquinone carries electrons from photosystem II to the cytochrome bf

complex; these electrons pass to the soluble protein plastocyanin, and

then to P700 to replace those lost during its photoexcitation. Electron

flow through the cytochrome bf complex is accompanied by proton pumping

across the thylakoid membrane, and the proton-motive force thus created

drives ATP synthesis by a CF0CF1 complex closely similar to the F0F1 complex

of mitochondria. This flow of electrons through photosystems II and I thus

produces both NADPH and ATP. A second type of electron flow (cyclic flow)

produces ATP only.



6 Both mitochondria and chloroplasts contain their own genomes and are

believed to have originated from prokaryotic endosymbionts of early

eukaryotic cells. Oxidative phosphorylation in aerobic bacteria and

photophosphorylation in photosynthetic bacteria are closely similar, in

machinery and mechanism, to the homologous processes in mitochondria and

chloroplasts.







f glucose yields much more energy than the fermentation pathways.

教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 脂代谢 授课方式及学时 讲授 8 学时





目的要求

目 1. 了解体内脂类的分布和生理功能：脂肪和类脂。

的 2. 了解脂类的消化与吸收。

要 3. 重点掌握脂肪动员，脂肪酸的氧化（β－氧化），酮体的生成、

求 氧化和生理意义。掌握甘油的代谢。

、 4. 了解甘油三酯的合成代谢及调节。

重 5. 了解磷脂代谢。

点 6. 掌握胆固醇合成原料、部位及代谢转化。

与 7. 掌握血脂的种类，重点掌握血浆脂蛋白的分类、组成与功能。了

难 解脂蛋白的代谢与高脂蛋白血症。

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理









1 Long-chain saturated fatty acids are synthesized from acetyl-CoA by a

cytosolic complex of six enzymes plus acyl carrier protein (ACP), which

contains phosphopantetheine as its prosthetic group. The fatty acid

synthase, which in some organisms consists of multifunctional

polypeptides, contains two types of -SH groups (one furnished by the

phosphopantetheine of ACP and the other by a Cys residue of the enzyme

a-ketoacyl-ACP synthase) that function as carriers of the fatty acyl

intermediates. Malonyl-ACP, formed from acetyl-CoA (shuttled out of

mitochondria) and CO2, condenses with an acetyl bound to the Cys -SH to

yield acetoacetyl-ACP with release of CO2. Reduction to the D-β-hydroxy

derivative and its dehydration to the trans-Δ2-unsaturated acyl-ACP is

followed by reduction to butyryl-ACP. For both reduction steps, NADPH is

the electron donor. Six more molecules of malonyl-ACP react successively

at the carboxyl end of the growing fatty acid chain to form palmitoyl-ACP,

the end product of the fatty acid synthase reaction. Free palmitate is

released by hydrolysis. Fatty acid synthesis is regulated at the level

of malonyl-CoA formation.



2 Palmitate may be elongated to yield the 18carbon stearate. Palmitate

and stearate in turn can be desaturated to yield palmitoleate and oleate,

respectively, by the action of mixed-function oxidases. Mammals cannot

make linoleate and must obtain it from plant sources. Mammals convert

exogenous linoleate into arachidonate, the parent compound of a family

of very potent hormonelike eicosanoids (prostaglandins, thromboxanes,

and leukotrienes).



3 Triacylglycerols are formed by reaction of two molecules of fatty

acyl-CoA with glycerol-3-phosphate to form phosphatidate, which is

dephosphorylated to a diacylglycerol then acylated by a third molecule

of fatty acyl-CoA to yield a triacylglycerol. This process is hormonally

regulated. Triacylglycerols are carried in the blood in chylomicrons.

Diacylglycerols are also the major precursors of glycerophospholipids.

In bacteria, phosphatidylserine is formed by the condensation of serine

with CDP-diacylglycerol, and decarboxylation of phosphatidylserine

produces phosphatidylethanolamine. Phosphatidylglycerol is formed by

condensation of CDP-diacylglycerol with glycerol-3phosphate followed by

removal of the phosphate in monoester linkage. Yeasts use similar pathways

in the synthesis of phosphatidylserine, phosphatidylethanolamine, and

phosphatidylglycerol; phosphatidylcholine is formed by methylation of

phosphatidylethanolamine. Mammalian cells have somewhat different

pathways for synthesizing phosphatidylcholine and

phosphatidylethanolamine. The head group alcohol (choline or

ethanolamine) is activated as the CDP-derivative, then condensed with

diacylglycerol. Phosphatidylserine is derived only from

phosphatidylethanolamine. The synthesis of plasmalogens involves

formation of their characteristic double bond by a mixedfunction oxidase.

The head groups of sphingolipids are attached by unique mechanisms.

Phospholipids are moved to their intracellular destinations by transport

vesicles or specific proteins.

4 Cholesterol is formed from acetyl-CoA in a complex series of reactions

through the intermediates β-hydroxy-β-methylglutaryl-CoA, mevalonate,

and two activated isoprenes, dimethylallyl pyrophosphate and isopentenyl

pyrophosphate. Condensation of isoprene units produces the noncyclic

squalene, which is cyclized to yield the steroid ring system and side chain.

Cholesterol synthesis is inhibited by elevated intracellular cholesterol.

Cholesterol and cholesteryl esters are carried in the blood as plasma

lipoproteins. Very low-density lipoprotein (VLDL) carries cholesterol,

cholesteryl esters, and triacylglycerols from the liver to other tissues,

where the triacylglycerols are degraded by lipoprotein lipase, converting

VLDL to low-density lipoprotein (LDL). The LDL, rich in cholesterol and

its esters, is taken up by receptor-mediated endocytosis, in which the

apolipoprotein B-100 of LDL is recognized by LDL receptors in the plasma

membrane. High-density lipoprotein (HDL) serves to remove cholesterol

from the blood, carrying it to the liver. Dietary conditions or genetic

defects in cholesterol metabolism may lead to atherosclerosis and heart

disease.



5 The steroid hormones (glucocorticoids, mineralocorticoids, and sex

hormones) are produced from cholesterol by alteration of the side chain

and the introduction of oxygen atoms into the steroid ring system. In

addition to cholesterol, a very wide variety of isoprenoid compounds are

derived from mevalonate through condensations of isopentenyl

pyrophosphate and dimethylallyl pyrophosphate. Prenylation of certain

proteins targets them for association with cell membranes and is essential

for their biological activity.



6 The fatty acid components of triacylglycerols furnish a large fraction

of the oxidative energy in animals. Triacylglycerols ingested in the diet

are emulsified in the small intestine by bile salts, hydrolyzed by

intestinal lipases, absorbed by intestinal epithelial cells and

reconverted into triacylglycerols, then formed into chylomicrons by

combination with specific apolipoproteins. Chylomicrons deliver

triacylglycerols to tissues, where lipoprotein lipase releases free fatty

acids for entry into cells. Triacylglycerols stored in adipose tissue of

vertebrate animals are mobilized by the action of hormones through a

hormone-sensitive triacylglycerol lipase. The fatty a,cids released by

this enzyme bind to serum albumin and are carried in the blood to the heart,

skeletal muscle, and other tissues that use fatty acids for fuel.



7 Once inside cells, free fatty acids are activated at the outer

mitochondrial membrane by esterification with coenzyme A to form fatty

acyl-CoA thioesters. These are converted into fatty acylcarnitine esters,

which are carried by a specific transporter across the inner mitochondrial

membrane into the matrix, where fatty acyl-CoA esters are formed again.

All subsequent steps in the oxidation of fatty acids take place in the

form of their coenzyme A thioesters, within the mitochondrial matrix.



8 In the first stage of fatty acid β oxidation, four reactions are

required to remove each acetyl-CoA unit from the carboxyl end of saturated

fatty acylCoAs: (1) dehydrogenation of the α and β carbons (C-2 and C-3)

by FAD-linked acyl-CoA dehydrogenases, (2) hydration of the resulting

trans-Δ2 double bond by enoyl-CoA hydratase, (3) dehydrogenation of the

resulting L-β-hydroxyacyl-CoA by NADlinked β-hydroxyacyl-CoA

dehydrogenase, and (4) CoA-requiring cleavage by thiolase of the

resulting β-ketoacyl-CoA to form acetyl-CoA and the coenzyme A thioester

of the original fatty acid, shortened by two carbons. The shortened fatty

acyl-CoA can then reenter the sequence, with loss of another acetyl-CoA.

For example, the 16-carbon palmitate yields altogether eight molecules

of acetyl-CoA, which in the second stage of fatty acid oxidation can be

oxidized to CO2 via the citric acid cycle. A large fraction of the

theoretical yield of free energy from fatty acid oxidation is recovered

as ATP by oxidative phosphorylation, the third and final stage of the

oxidative pathway.



9 Oxidation of unsaturated fatty acids requires the action of two

additional enzymes: enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase.

Oddcarbon fatty acids are oxidized by the same path

way but yield one molecule of propionyl-CoA. The latter is carboxylated

to methylmalonyl-CoA, which is isomerized to succinyl-CoA by a reaction

catalyzed by methylmalonyl-CoA mutase. This enzyme requires coenzyme Bl2,

a complex cofactor containing a cobalt ion in a corrin ring system.

Coenzyme B12 is involved in a number of enzymecatalyzed reactions in which

a hydrogen atom is exchanged with a functional group attached to an

adjacent carbon.



10 Fatty acid oxidation is tightly regulated. High carbohydrate intake

suppresses fatty acid oxidation in favor of fatty acid biosynthesis.



11 The ketone bodies acetoacetate, D-β-hydroxybutyrate, and acetone are

formed in the liver and are carried to other tissues, where they serve

as fuel molecules, being oxidized to acetyl-CoA and thus entering the

citric acid cycle. The overproduction of ketone bodies in uncontrolled

diabetes or severe starvation can lead to acidosis or ketosis.





教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次

授课内容 RNA 的生物合成--转录 授课方式及学时 讲授 8 学时





目的要求

目 1、重点掌握转录的原料、模板、酶和因子。

的 2、掌握转录的基本过程。

要 3、了解转录后的加工修饰。

求

、

重

点

与

难

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 Instant notes of Biochemistry 2nd edition

书 2 生物化学 第 6 版

目 3 LEHNINGER 生物化学原理









1 Transcription is catalyzed by DNA-directed RNA polymerase, a complex

enzyme that synthesizes RNA complementary to a segment of one strand (the

template strand) of duplex DNA, starting from ribonucleoside

5'-triphosphates. To initiate transcription, RNA polymerase binds to a

DNA site called a promoter. Bacterial RNA polymerase requires a special

subunit for recognizing the promoter. As the first committed step in

transcription, binding of RNA polymerase to promoters is subject to many

forms of regulation. Eukaryotic cells have three different types of RNA

polymerases. 'I~anscription stops at specific sequences called

terminators. Many copies of an RNA chain can be transcribed simultaneously

from a single gene.



2 Ribosomal RNAs and transfer RNAs are made from longer precursor RNAs

that are trimmed by nucleases, and some bases are modified enzymatically

to yield the mature RNAs. In eukaryotes, messenger RNAs are also formed

from longer precursors. Primary RNA transcripts often contain noncoding

regions called introns, which are removed by splicing. Group I introns

are found in rRNAs and their excision requires a guanosine cofactor. Some

group I and some group II introns are capable of self-splicing; no protein

enzymes are required. Nuclear mRNA precursors have a third class of

introns that are spliced with the aid of RNA-protein complexes called

snRNPs. The fourth class of introns, found in some tRNAs, are the only

ones known to be spliced by protein enzymes. Messenger RNAs are also

modified by addition of a 7-methylguanosine residue at the 5' end, and

cleavage and polyadenylation at the 3' end to form a long poly(A) tail.



3 The self splicing introns and the RNA component of RNase P (the enzyme

that cleaves the 5' end of tRNA precursors) form a new class of biological

catalysts called ribozymes. These have the properties of true enzymes and

are effective catalysts. They promote two types of reaction, hydrolytic

cleavage and transesterification, using RNA as substrate. Combinations

of these reactions are promoted by the excised group I rRNA intron from

Tetrahymena, resulting in a type of RNA polymerization reaction. The study

of these reactions and of introns themselves has provided insights into

likely pathways for biochemical evolution.



4 Polynucleotide phosphorylase can reversibly form RNA-like polymers from

ribonucleoside 5'diphosphates, adding or removing ribonucleotides at the

3'-hydroxyl end of the polymer. It acts in vivo to degrade RNA.



5 RNA-directed DNA polymerases, also called reverse transcriptases, are

produced in animal cells infected by RNA viruses called retroviruses.

These enzymes transcribe the viral RNA into DNA. This process can be used

experimentally to form complementary DNA. Many eukaryotic transposons are

related to retroviruses, and their mechanism of transposition includes

an RNA intermediate. The enzyme that synthesizes telomeres, called

telomerase, is a specialized reverse transcriptase that contains an

internal RNA template.



6 RNA-directed RNA polymerases, or replicases, are found in bacterial

cells infected with certain RNA viruses. They are template-specific for

the viral RNA.

7 The existence of catalytic RNAs and pathways for the interconversion

of RNA and DNA has led to speculation that the earliest living things were

made up entirely or largely of RNA molecules that served both for

information storage and for catalysis of replication.







教师： 授课时间：

课 程 生物化学 课次 4 授课专业及班次



授课内容 DNA 复制 授课方式及学时 讲授 8 学时





目的要求

目 1. 掌握 DNA 复制的方式――半保留复制。重点掌握复制的原料、

的 模板及参与复制的酶类和因子。掌握复制的基本过程。

要 2. 掌握 DNA 的损伤与修复的概念及方式。

求 3. 掌握逆转录的过程。

、

重

点

与

难

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参 1 Instant notes of Biochemistry 2nd edition

考 2 生物化学 第 6 版

书 3 LEHNINGER 生物化学原理

目

1 The integrity of the structure and nucleotide sequence of DNA is of

utmost importance to the cell. This is reflected in the complexity and

redundancy of the enzyme systems that participate in DNA replication,

repair, and recombination.



2 Replication of DNA occurs with very high fidelity and within a designated

time period in the cell cycle. Replication is semiconservative, with each

strand acting as a template for a new daughter strand. The reaction starts

at a sequence in the DNA called the origin, and usually proceeds

bidirectionally from that point. DNA is synthesized in the 5'?3' direction

by DNA polymerases. At the replication fork, the leading strand is

synthesized continuously and in the same direction as replication fork

movement. The lagging strand is synthesized discontinuously. The fidelity

of DNA replication is maintained by (1) base selection by the polymerase,

(2) a 3'?5' proofreading exonuclease activity that is part of most DNA

polymerases, and (3) a specific repair system that repairs any mismatches

left behind after replication.



3 Most cells have several DNA polymerases. In E. coli, DNA polymerase III

is the primary replication enzyme. DNA polymerase I is responsible for

special functions during replication, recombination, and repair. DNA

polymerase II has a specialized replication activity that allows it to

replicate past DNA lesions in error-prone DNA repair. Replication of the

E. coli chromosome involves many enzymes and protein factors organized

into complexes. Initiation of replication requires binding of DnaA

protein to the origin, strand separation, and the entry of the DnaB and

DnaC proteins to set up two replication forks. The action of DnaA is

associated with the E. coli membrane and is regulated by the action of

acidic phospholipids. Initiation is the only phase of replication that

is regulated. The process of elongation has different requirements for

each strand. DNA strands are separated by helicases, and the resulting

topological strain is relieved by topoisomerases. Single-strand DNA

binding proteins stabilize the separated strands. In synthesis of the

lagging strand, the primosome protein complex moves with the fork and

regulates the synthesis of RNA primers by primase. Synthesis of the

leading and lagging strands by DNA polymerase III may be coupled. RNA

primers are removed and replaced with DNA by DNA polymerase I, and nicks

are sealed by DNA ligase.



4 A similar pattern of replication occurs in eukaryotic cells, but

eukaryotic chromosomes have multiple replication origins. Several

eukaryotic DNA polymerases have been identified.



5 Every cell also has multiple and sometimes redundant systems for DNA

repair. Mismatch repair in E. coli is directed by transient

undermethylation of (5')GATC sequences on the newly synthesized strand

after replication. Other systems recognize and repair damage caused by

environmental agents such as radiation and alkylating agents, and damage

caused by spontaneous reactions of nucleotides. Some repair systems

recognize and excise only damaged or incorrect bases te.g., uracil),

leaving an AP (apurinic or apyrimidinic) site in the DNA. This is repaired

by excising and replacing the segment of DNA containing the AP site. Other

excision repair systems recognize and remove pyrimidine dimers and other

modified nucleotides. Some types of DNA damage can also be repaired by

direct reversal of the reaction causing the damage: pyrimidine dimers are

directly converted to monomeric pyrimidines by photolyase, and the methyl

group in O6-methylguanine is removed by a specific methyltransferase.

Errorprone repair is a specialized and mutagenic replication process

observed when DNA damage is so heavy that the need for some replication

outweighs the need to avoid errors.



6 DNA sequences are rearranged in recombination reactions. Homologous

genetic recombination occurs between any two DNAs that share sequence

homology. This reaction takes place in meiosis (in eukaryotes) and is one

of the processes that creates genetic diversity. Homologous recombination

also is needed for repair of some types of DNA damage. A Holliday

intermediate in which a crossover has occurred between the strands of two

homologous DNAs is formed during the process. In E. coli, the RecA protein

promotes formation of Holliday intermediates and branch migration to

extend heteroduplex DNA.



7 Site-specific recombination occurs only at specific target sequences

and can also involve a Holliday intermediate. The recombinases cleave the

DNA at specific points and ligate the strands to new partners. This type

of recombination is found in virtually all cells, and its many functions

include DNA integration and regulation of gene expression. In vertebrates,

a programmed recombination reaction related to site-specific

recombination is used to join immunoglobulin gene segments to form

immunoglobulin genes during B-lymphocyte differentiation. Some small

segments of DNA, called transposons, are capable of moving from one point

in a chromosome to another point in the same or another chromosome. These

elements are found in virtually all cells.







教师： 授课时间：

课 程 分子生物学 课次 4 授课专业及班次 本科生物技术专业



授课内容 基因表达的调控 授课方式及学时 讲授 8 学时

目的：

目

的 目的要求： 使学生掌握原核转录调控的机制

要

求 教学内容： 半乳糖操纵子、色氨酸操纵子、其他 s 因子对转录的调控等

、

目的要求： 使学生掌握真核转录调控的机制

重

教学内容： 真核转录因子，转录调控的实例

点

与

难

重点：

点

1. 掌握核酸的分类、细胞内分布及生物学功能。

2、了解核酸的元素组成，平均磷含量及换算。

3、掌握核苷酸、核苷、碱基的基本概念及其结构。

4、掌握核酸的紫外吸收特性，核酸变性、复性及杂交等概念

难点：

DNA 的三级结构――核小体。三类 RNA――rRNA、mRNA、tRNA

的结构特点及功能



课

1.板书与挂图相结合。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 生物化学 第 6 版

书

目

2 LEHNINGER 生物化学原理









Gene and Operon







A "gene"

The entire nucleic acid sequence that is necessary for the synthesis of

a functional polypeptide or RNA molecule.



 Thus, a gene contains additional sequence information beyond that

which codes for the amino acids in a protein or the nucleotides in

an RNA molecule.

 The gene also contains the DNA necessary to get a particular

transcript made.



Note: Transcription control regions can be remote to the coding region

(on the order of Kb's or 10's of Kb's away).



 Most prokaryotic genes lack introns (intervening DNA sequence).

 In prokaryotes, genes which encode proteins with relationships in

a metabolic pathway form Operons - which produce polycistronic

mRNA's.



Definition of "Operon"



In bacterial DNA, a cluster of contiguous genes transcribed from one

promoter that gives rise to a polycistronic mRNA.



Definition of "Promoter"



A DNA sequence to which RNA polymerase binds prior to initiation of

transcription - usually found just upstream of the transcription start

site of a gene



e.g. Trp Operon - involved in the biosynthesis of the amino acid

tryptophan:









 A consequence of the arrangement of bacteria genes into operons is

that the level of mRNA for each of the genes in the operon is exactly

the same.



Note: ribosomes transcribe from the start of each gene, not only from the

first gene.



 Another consequence of the arrangement of bacteria genes into

operons is that an upstream mutation (i.e. possibly inhibiting

transcription) can prevent "downstream" genes from being

transcribed and expressed.

 Most eukaryotic transcription units produce monocistronic mRNA's,

i.e. they encode only one protein.

 There is a fundamental difference in the translation processes of

prokaryotes and eukaryotes:



1. In prokaryotes ribosomes can bind at specific recognition sequences

anywhere within the mRNA (called ribosome binding sites, or

"Shine-Dalgarno" sites).

2. In eukaryotes, ribosomes bind via the interaction with specifically

modified 5' region (so called 5' cap site) of mRNA molecules.

3. Most eukaryotic mRNA's are therefore monocistronic.



 Mutations in simple eukaryotic transcription units affect only one

protein.



Complex Eukaryotic Transcription Units



 The primary RNA transcript encoded by complex transcription units

can be spliced in more than one way.

 Because of the different processing possibilities, the exons

(coding regions) in a single complex transcription unit can be

linked in alternative ways, to yield different mRNAs and different

proteins.







Transcriptional regulation



Successful survival requires adaptability and economy:



1. The ability to switch from metabolizing one substrate to another

as environmental resources change

2. It would be an energetic waste to produce enzymes for a metabolic

pathway which is not needed.



Induction versus Repression of Enzyme Synthesis



 In E. coli certain enzymes are produced only when the cells are grown

on certain substrates. This effect is called enzyme induction.

 For example, when cells are grown in the absence of a type of sugar

known as a galactoside (e.g. lactose) the cells contain very few

molecules (~5 per cell) of the enzyme -galactosidase (which

cleaves lactose into glucose and galactose).

o There is no need for this enzyme in the absence of lactose.

o If lactose is added to E. coli, in a very short amount of time

there are approximately 5000 molecules of -galactosidase

per cell (approximately ~1,000 fold induction).

o If lactose is removed from the media synthesis of

-galactosidase stops.

 A similar but opposite situation occurs in regard to the synthesis

of tryptophan (the biosynthetic enzymes are contained in the trp

operon).

o In this case production of the enzymes for tryptophan

biosynthesis are rapidly shut down if tryptophan is present,

in a process called repression.



 Repression is a transcriptional regulatory mechanism for commonly

required gene products

 Induction is a transcriptional regulatory mechanism for gene

products which may be required under unusual or infrequent

situations







1998 Dr. Michael Blaber



BCH5425 Molecular Biology and Biotechnology

Spring 1998

Dr. Michael Blaber

blaber@sb.fsu.edu





Lecture 16

The lac Operon, CAP Site





The E. coli lac operon







 lac Z codes for -galactosidase, which is an enzyme that cleaves

-galactosides (e.g. lactose).

 lac Y codes for permease, which is involved in the transport of

-galactosides into the cell.

 lac A codes for -galactoside transacetylase, which acetylates

-galactosides.



 A mutation in either lac Z or lac Y can lead to a lac- genotype,

i.e. cells which cannot utilize -galactosides as a nutrient.

 A lac A- mutant, lacking transacetylase activity, can still utilize

-galactosides (it is still lac+ genotype). Its role in the

metabolism of galactosides is not clear.



Promoter



a region of DNA involved in binding of RNA polymerase to initiate

transcription.



Terminator



a sequence of DNA that causes RNA polymerase to terminate transcription.



 The cluster of three genes, lac ZYA, is transcribed into a single

mRNA (polycistronic message) from a promoter just upstream from the

lac Z gene.

 In the absence of an inducer the gene cluster is not transcribed.

 When an inducer is added (e.g. lactose, or the non-hydrolyzable

analog isopropyl thiogalactoside - IPTG) transcription starts at

a single promoter (lac P) and proceeds through the lac ZYA genes

to a terminator sequence located downstream of the lac A gene.



Note: The lac ZYA mRNA has a half life of ~3 minutes, which allows induction

to be reversed relatively rapidly (i.e. cells stop producing enzymes

rapidly after induction stops).



What molecule does the inducer (lactose) interact with to affect

transcriptional regulation (i.e. induction of the lac operon)?



Answer: it is not -galactosidase, permase or transacetylase, rather it

is a separate protein called a repressor protein.



 The lac genes are controlled by a mechanism called negative

regulation.

o This means that they are transcribed unless they are turned

off by the regulator protein.

o A mutation that inactivates the regulator protein causes the

lacZYA genes to be continually expressed.



Since the function of the regulator protein is to prevent expression, it

its called a repressor protein.



There are two types of genes in the lac operon:



1. Structural genes - they code for enzymes required for some

biochemical pathway (e.g. lac Z, Y and A).

2. Regulator genes - they code for proteins involved in regulation of

structural genes.



Mutations in structural genes typically affect the function of only that

structural gene.

Mutations in regulator genes can affect the expression of all structural

genes in an operon.



lac I is the regulator gene of the lac operon.



 This gene is located just upstream of the promoter region for the

lac structural genes.

 The lac I gene has its own promoter (constitutive) and terminator.

 It makes a monocistronic message, and codes for one protein - the

lac repressor protein.



The crucial feature of the lac "control circuit" resides in the dual

feature of the lac I repressor protein:



1. It can prevent transcription

2. It can recognize and bind the small molecule inducer (lactose or

IPTG)



Prevention of transcription by the lac repressor



 lac repressor (active as a tetrameric protein) binds to a sequence

of DNA called the operator (lac O region).

o The operator region lies between the lac promoter region

(site of RNA polymerase binding and transcription initiation)

and the lac Z gene.

o The first 26 base pairs of the lac Z gene comprise the operator

region.



When the repressor binds to the operator region, its presence prevents

RNA polymerase from initiating transcription at the promoter.



 It is not that the repressor protein "blocks" the movement of RNA

polymerase through the lac Z gene.

 Repressor binding and RNA polymerase binding (to the promoter) are

mutually exclusive at the lac promoter/operator (lac PO) region.



How does the repressor/operator interaction change in the presence of the

inducer molecule?

 The inducer can bind to the repressor to form a repressor/inducer

complex that no longer associates with the operator.

o The key feature of this interaction is that the repressor

protein has two binding sites, one for the inducer and one

for the operator.

o When the inducer binds at its site, it changes the

conformation of the repressor protein such that the operator

binding site has a much reduced affinity for the DNA operator

region.

o This type of control is called allosteric control.

o The result is that when the inducer is added the repressor

is converted to a form which releases from the operator.







Positive control of the lac operon is exerted by cAMP-CAP complex



 E. coli prefers glucose over other carbon sources.



When glucose enters an E. coli cell it is utilized directly without

induction of any new enzymes.



 When E. coli is grown on glucose, if another sugar (e.g. lactose)

is added the induction of enzymes to utilize the other sugar does

not occur until the glucose is used up.



 When E. coli is starved for glucose it synthesizes an unusual

nucleotide: cyclic 3'5' adenosine monophosphate (cyclic AMP, or

cAMP):







1. In bacteria an increase in the cAMP level seems to be an "alert"

signal indicating a low glucose level:







Dibutyryl cAMP



 an analogue of cAMP which can pass through the E. coli membrane and

into the cell.

 If this is added to media containing glucose and lactose it will

result in the induction of the lac operon.

 Thus, it mimics the chemical message which tricks the E. coli to

respond as though glucose levels were low.

 Mutants of E. coli have been isolated which cannot be induced to

metabolize any sugar other than glucose. There were two general

categories of mutants:



1. Class I. Defective in the enzyme adenylate cyclase. These mutants

are unable to make cAMP even when the glucose conentration is low.

2. Class II. Lacks a particular protein known as cAMP receptor protein

(CRP) or, also known as catabolite receptor protein (CRP).



 Maximum transcription from the lac operon requires the presence of

a cAMP/CRP complex.

o cAMP/CRP complex binds to a specific sequence in the lac

control region called the "CAP" site.

o The CAP site is just upstream from the RNA polymerase binding

site.

o Mutations in the CAP site that prevent cAMP-CRP binding also

prevent high levels of expression of the lac operon.

 Thus, bound cAMP/CRP complex activates transcription (positive

control), whereas bound lac repressor inhibits transcription

(negative control).

o cAMP/CRP complex has affinity for DNA, and RNA pol.

o Enhances complex formation of RNA pol with the DNA promoter

region.







1998 Dr. Michael Blaber



BCH5425 Molecular Biology and Biotechnology

Spring 1998

Dr. Michael Blaber

blaber@sb.fsu.edu





Lecture 17

Transcriptional Regulation: the lac operon, cont., DNA footprinting





Induction of the lac operon with lactose analogues



 The lac operon can be induced with lactose

o -galactosidase (lacZ gene product) metabolizes the lactose

o When levels of lactose are reduced, the lac operon is again

repressed by the lac repressor (lacI gene product)

 Non-metabolized lactose analogues can continually induce (i.e.

de-repress) the lac operon

o isopropyl -thiogalactoside, or IPTG, is a non-metabolized

lactose analogue



DNA "footprinting" experiments



 If a protein binds to a region of DNA, it can protect that region

of DNA from digestion by dnase (DNAse I: an endonuclease at sites

adjacent to pyrimidine nucleotides).

32

o A fragment of DNA can be labeled at the 5' ends with P and

then the label can be preferentially removed from one end (i.e.

the 3' end of a gene) by an appropriate restriction

endonuclease.

o If this DNA fragment, with a label at one specific end, forms

a complex with a DNA binding protein the protein will protect

the region of DNA that it binds to from DNAse I digestion.

o The digestion is done so as to be incomplete, for the purposes

of this discussion, imagine that each DNA molecule is cleaved

only once. Furthermore, the site of cleavage is randomly

chosen from the available sites.

o Fragments of the DNA, separated and analyzed by size (using

gel electrophoresis) after digestion will indicate the

protected region:









Results of footprinting experiments



lac DNA incubated with either cAMP/CAP protein, or RNA polymerase, or lac

I repressor protein:







 RNA polymerase interacts with specific promoter sequences and

produces a "footprint" over a region of ~70 base pairs.

o This protection was observed to be more obvious on one strand

than the other (i.e. if the other strand was labeled the

results did not show as much protection).

o This region of DNAse protection included sites in the DNA from

which mutagenesis experiments produced either "up"

regulation or "down" regulation of promoter strength.

o These mutagenic "hot" spots affecting promoter strength were

located at positions either -10 or -30 upstream from the

transcription start site (position +1 in the above diagram):







 Promoters can be classified according to their "strength".

 This refers to the relative frequency of transcription initiation

(transcriptional initiation events per minute), and is related to

the affinity of RNA polymerase for the promoter region .

 Many promoters in E. coli have been characterized and a "consensus"

promoter sequence has been identified:





Note: The lac promoter is a relatively weak promoter.



RNA Polymerase



 E. coli RNA polymerase is a holoenzyme comprised of subunits '

(dimer) and 70.

o The  subunit is the subunit which binds to the promoter

70





region, but is unable to initiate RNA synthesis.

o After the  subunit subunit binds, the other subunits bind

70





forming a function RNA polymerase.

o After approximately 10 base pairs have been transcribed the

70 subunit leaves and the core polymerase continues on.



The lac Operator Region



 The lac operator region is comprised of an (imperfect) inverted

repeat region.

 Not surprisingly active repressor molecules are composed of a

homodimer.

o In the homodimer structure there are a pair of -helix

regions which insert into adjacent major grooves of the DNA.

o The separation is approximately 34 angstroms apart.









1998 Dr. Michael Blaber



BCH5425 Molecular Biology and Biotechnology

Spring 1998

Dr. Michael Blaber

blaber@sb.fsu.edu

Lecture 18

Transcriptional Regulation: Transcription termination, the trp operon





Transcription termination in prokaryotes



 Because protein coding regions are closely spaced in the genomes

of microorganisms, independent control of neighboring genes is

possible only if a transcription termination site lies between them

(i.e. downstream genes must be independently regulated).

 There are two general mechanisms of transcription termination:



1. one requires the presence of a transcription termination protein

called rho,

2. the other requires no associated proteins.



Rho-independent termination



 Rho-independent transcription termination sequences have two

general characteristic features:



1. A 3' stretch of T residues

2. A 'GC' rich interrupted palindrome just upstream of the 3' poly T

region.



The important features of Rho-independent transcription termination are

as follows:



1. The inverted repeat region can self-hybridize to form a "stem-loop"

structure:

2. The GC rich stem loop structure interacts with RNA polymerase to

cause it to pause.

3. The short UUU region, which is base pairing with AAA sequence on

the anti-sense DNA strand, has low thermal stability and melts -

releasing the nascent RNA transcript.



Attenuation at the trp operon and premature mRNA chain termination



 The trp operon is regulated by the trp repressor protein.

o Trp repressor protein binds to tryptophan and undergoes a

conformational change which allows it to bind to the trp

operator region (downstream of the promoter).

o This prevents transcription of the trp operon in the presence

of tryptophan (i.e. tryptophan represses expression).

o However the repression is somewhat incomplete and there is

a second mechanism which contributes to transcription

repression.

 When tryptophan is present, and the operon is repressed, the few

transcripts which are made are actually quite short and do not

encompass the entire polycistronic message of the trp operon.

 The short mRNA that is made comprises only a region called the leader

sequence:







 The leader region contains an attenuator sequence - a site where

a choice is made between elongation of the growing trp transcript

or (premature) termination.



 Attenuation depends on the interplay between ribosome binding and

translation of the nascent mRNA transcript and the formation of a

particular stem-loop structure in the mRNA leader sequence.

o Formation of this structure depends on the rate of ribosomal

translation of the leader sequence (the leader sequence

contains an AUG start codon).

o Efficient translation of the leader sequence depends upon the

concentration of charged tRNA's for the appropriate amino

acids coded for by the mRNA leader sequence.

 The leader sequence has the following characteristics:



1. It contains a ribosome binding site and an AUG start codon necessary

for the initiation of translation.

2. The corresponding amino acid sequence coded for by the leader

sequence contains several tryptophan residues.

3. It contains several inverted repeat sequences such that the mRNA

can adopt different alternative structures.

4. One of these structures represents a rho-independent termination

site.







Under conditions of low tryptophan



 A ribosome translating the nascent trp polycistronic mRNA stalls

at the codons for tryptophan in the leader mRNA sequence.

o This prevents segments 1 and 2 of the nascent mRNA from

forming a potential stem-loop structure, and frees up segment

2 to form a stem loop structure with segment 3.

o In this case, nascent transcription can proceed through,

transcribing the entire trp operon.



Under conditions of high tryptophan



 A ribosome translating the nascent trp polycistronic mRNA does not

stall at segment 1 (the region with codons coding for tryptophan).

o In this case the stem-loop structure between 1 and 2 is not

prevented from forming, and thus the stem-loop structure 3-4

can form.

o This stem loop structure is essentially a rho-independent

transcription termination structure.

o The RNA polymerase transcribing the nascent message will

terminate.



Rho-dependent transcription termination



 About half the termination sites in E. coli require an accessory

protein called the rho factor.

 Many rho-dependent sites have been characterized from E. coli and

no obvious sequence similarity is present.

 Rho associates with the nascent RNA transcript and this interaction

activates an ATPase activity which appears to allow rho to

translocate along the mRNA in the 3' direction.

o It may be that if the RNA polymerase pauses, rho can catch

up and cause termination (i.e. bump RNA polymerase off the

DNA template?).



Pausing of RNA polymerase is thought to be an important mechanism of

rho-dependent transcription termination.



教师： 授课时间：

课 程 课次 4 授课专业及班次



授课内容 DNA 授课方式及学时 讲授 8 学时

目的要求

目

的 1.第三章 核酸性质

要 目的要求： 介绍核酸的一些基本性质，为了解遗传信息遗传、基因表达与调控和

求 掌握 DNA 技术做铺垫。

、

重 教学内容： 核酸结构、核酸的化学和物理性质、核酸的光谱和热性质、DNA 超螺

点 旋

与

难

点









课

1.多媒体教学。

堂

。

设

2.重点内容进行归纳。

计









参

考 1 生物化学 第 6 版

书

目

2 LEHNINGER 生物化学原理









DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are composed of

two different classes of nitrogen containing bases: the purines and

pyrimidines. The most commonly occurring purines in DNA are adenine and

guanine:







The most commonly occurring pyrimidines in DNA are cytosine and thymine:

RNA contains the same bases as DNA with the exception of thymine. Instead,

RNA contains the pyrimidine uracil:







Adenine, guanine, cytosine, thymine and uracil are usually abreviated

using the single letter codes A, G, C, T and U, respectively.



Purines and pyrimidines can form chemical linkages with pentose (5-carbon)

sugars. The carbon atoms on the sugars are designated 1', 2', 3', 4' and

5'. It is the 1' carbon of the sugar that becomes bonded to the nitrogen

atom at position N1 of a pyrimidine or N9 of a purine. DNA precursors

contain the pentose deoxyribose. RNA precursors contain the pentose

ribose (which contains an additional OH group at the 2' position):







The resulting molecules are called nucleosides and can serve as elementary

precursors for DNA and RNA synthesis, in vivo.



Before a nucleoside can become part of a DNA or RNA molecule it must become

complexed with a phosphate group to form a nucleotide (either a

deoxyribonucleotide or ribonucleotide). Nucleotides can posess 1, 2 or

3 phosphate groups, e.g. the nucleotides adenosine monophosphate (AMP),

adenoside diphosphate (ADP) and adenosine triphosphate (ATP). The

phosphate groups are attached to the 5' carbon of the ribose sugar moiety.

Beginning with the phosphate group attached to the 5' ribose carbon, they

are labeled ,  and  phosphate. It is the tri-phosphate nucleotide which

is incorporated into DNA or RNA.







DNA and RNA are simply long polymers of nucleotides called polynucleotides.

Only the  phosphate is included in the polymer. It becomes chemically

bonded to the 3' carbon of the sugar moiety of another nucleotide:







In other words, the polynucleotide is connected by a series of 5' to 3'

phosphate linkages. Note the sequence of the bases in the above diagram.

Polynucleotide sequences are referenced in the 5' to 3' direction.

Typically, polynucleotides will contain a 5' phosphate and 3' hydroxyl

terminal groups. The common representation of polynucleotides is as an

arrow with the 5' end at the left and the 3' end at the right.

Summary of terms:



RNA DNA

Base Nucleoside Nucleotide Code

(monophosphate) (monophosphate)

(Adenylic

Adenine Adenosine AMP dAMP A

acid)

(Guanylic

Guanine Guanosine GMP dGMP G

acid)

(Cytidylic

Cytosine Cytidine CMP dCMP C

acid)

(Thymidylic

Thymine Thymidine dTMP T

acid)

(Uridylic

Uracil Uridine UMP U

acid)



What is the structure of DNA? How is the structure related to function?



1950's



The primary chemical structure of polynucleotides was known (i.e. the

3'-5' phosphate linkage).



1951 E. Chargaff



The experiment:



Take DNA from a variety of species and hydrolyze it to yield individual

pyrimidines and purines. Determine the relative concentrations of the A,

T, C and G bases.



Result:



Although different species had uniquely different ratios of pyrimidines

or purines, the relative concentrations of adenine always equaled that

of thymine, and guanine equaled cytosine.



Chargaff's Law: A=T, G=C



1950's R.E. Franklin



X-ray diffraction studies of DNA fibers demonstrated that DNA adopted a

highly ordered helical structure. Franklin concluded that two or more

chains must coil around each other to form a helix. Some basic dimensions

of the helix were calculated from the x-ray diffraction data.



1953 L. Pauling and R.B. Corey



Propose a three chain helical structure for DNA with the phosphate

backbone in the center and the bases on the outside.



1953 J.D. Watson and F.H.C. Crick



Identified a hydrogen bonding arrangement between models of thymine and

adenine bases, and between cytosine and guanine bases which fullfilled

Chargaff's rule:







Note that the "TA" pair can overlay the "GC" pair with the bonds to the

sugar groups in similar juxtaposition. In the "double helix" model of

Watson and Crick the polynucleotide chains interact to form a double helix

with the chains running in opposite directions. The bases are directed

towards the center (and stack on top of one another) and the sugar

backbones face the outside of the helix.



The Watson and Crick model had the following physical dimensions:



 34 Å per helical repeat

 10 base pairs per repeat (i.e. per turn of the helix)

 3.4 Å inter-base stacking distance

 20 Å diameter for the helical width



Physical characteristics of the model matched those determined by

Rosalind Franklin's x-ray diffraction studies.



Consequenses of the model for genetic information:



The Watson and Crick paper was an exercise in brevity (1 page only in

Nature). The structure was so rich with implication that quite a bit could

be written. The authors, however, chose only to say "It has not escaped

our notice that the specific pairing we have postulated immediately

suggests a possible copying mechanism for the genetic material".



1. If G always paired with C, and T always paired with A, then either

strand could be regenerated from the complementary information in

the other strand.

2. The basis of the complementarity was hydrogen bonding, i.e.

non-covalent interactions which could be easily broken and

re-formed.

3. The information which DNA carried was within the unique base

sequence of the DNA.

4. From the general interior location of the bases, it would appear

that the double helix would have to dissociate in order to access

the information.

5. The non-equitorial location of the sugar moieties (see above)

suggested that the DNA helix would have a major groove and a minor

groove.







Lecture 7



DNA Supercoiling







Unwinding of the helix during DNA replication (by the action of helicase)

results in supercoiling of the DNA ahead of the replication fork.



 This supercoiling increases with the progression of the replication

fork.

 If the supercoiling is not relieved, it will physically prevent the

movement of helicase.



The topology of DNA can be described by three parameters:



1. Linking Number (L) An integer value. "Positive" is referenced as

right-handed.

2. Twist (T) A real number (the "apparent" linkage number)

3. Writhe (W) A real number ("supercoils" in the DNA structure)



Consider closed circular DNA:



 Linking number is an integer value.

 It refers to the number of times the two strands of the duplex make

a complete 360 degree turn.



For circularly closed DNA, like the E. coli genome, the linking number

can only be changed if we do the following:



1. physically break the duplex

2. introduce (or remove) a 360 degree turn

3. ligate (covalently close) the break.







Rubber tubing "helix" experiment



Cut two lengths of 1/8" rubber tubing, each about 20" long. Insert a

smaller piece of tubing, or piece of pipette tip in the ends to allow the

ends to be connected. These two pieces of rubber tubing represent each

strand of a DNA duplex. DNA can be ligated, or joined, when we have 5'

(phosphate) and 3' (hydroxyl) ends. So we need a way to keep track of which

end is which for each piece of tubing.



1. You can either write "5" and "3" on opposite ends of each piece and

align them in opposite directions, or

2. On one piece of tubing, mark both ends with a sharpie. Only similarly

colored ends can be ligated (see diagram above)



This will allow you to maintain correct strand "orientation" when you

"ligate" the strands of the duplex.



 Introduce a Linking number = +2 (two 360° right handed twists into

the duplex)

 then "ligate" the ends (make sure you maintain strand orientation,

i.e. you connect the appropriate two strands).



Confirm that the correct linking number has been introduced by "melting"

the duplex on one side and forcing all turns into a small region of the

duplex (easy to count this way).



 Confirm that looking down the helix at the turns that they are "right

handed" (does not matter which way you look down the helix).



Note that the "duplex" when held between thumb and forefinger and allowed

to hang, prefers a "supercoiled" topology, as opposed to "relaxed" [Note:

this is usually seen with very skinny tubing, larger diameter tubing may

not readily adopt this topology].



 Confirm that the "supercoils" are actually left handed as you look

down the supercoils (regardless of direction down the supercoils).

 The "supercoil" is most likely a full 360°, rather than 180°. In

any case, hold the ends of the duplex so that a left handed 360°

"supercoil" is present.

 Now, count how many times the strands of the "duplex" cross each

other. In this conformation the strands of the "duplex" will not

actually cross each other (Note: you may have one strand crossing

and then later uncrossing, for a net result of no crossing).



Thus, in response to the introduction of +2 Linking number, the "duplex"

can adopt +2 (180°) "supercoils", such that the resulting apparent

linkage number (i.e. "twist" value) of the two strands is zero



(Note: that a supercoil is considered positive, if it is "left handed").



 Remove one "positive" supercoil by unwinding by 180°.

 Now hold the ends of the "duplex" and count the apparent linkage

number (i.e. "twists"). There will be a single right handed "twist".

 Thus, a single 180° "positive" supercoil has the effect of

removing a single "positive" twist (i.e. reduces the apparent

linkage number by 1).



The Writhe number refers to the number of supercoils present.



 Although it may seem that the consequence of introducing

supercoiling (Writhe) is changing the Linking number, it is not.

 The consequence of Writhe is that the Twist (apparent linkage

number) is altered (increased or decreased).



DNA has a preferred "Twist" value (preferred apparent linkage number) for

a specified length of DNA:



 Watson and Crick's model of the DNA duplex had 10 basepairs per turn.

 Under physiological conditions of salt (0.15 M NaCl) and

temperature, DNA prefers to adopt about 10.6 bp/turn .

 Writhe is introduced in the DNA to achieve this value for the

"Twist" (apparent linkage number)



For a given (fixed) Linkage number over a given length of DNA, the DNA

can adopt either positive or negative supercoils to achieve a "twist"

(apparent linkage number) such that there will be 10.6 basepairs/turn.



Linkage number does not change with supercoiling (it can only change by

breaking the duplex)



 Writhe has the effect of changing the apparent Linkage number.

 One supercoil is defined as being able to change the apparent

linkage number by +/- 1.



The twist value (apparent linkage number) for a given length of DNA is

related to the number of base pairs per turn that the DNA wants to adopt:

Linkage Number = (size of DNA in base pairs)/(basepairs/turn) + Writhe



or



Linkage Number = #of Twists + Writhe



this is usually abbreviated as



Linkage = Twist + Writhe



L = T + W







For example, if we have a circularly closed DNA molecule with a length

of 5300 base pairs, and a preferred conformation of 10.6 basepairs per

turn, can it achieve this conformation without having to introduce any

supercoiling (i.e. writhe)?



Apparent linkage number (Twist) = (5300 base pairs) / (10.6 base

pairs/turn)



Apparent linkage number (Twist) = 500



In other words, in order to achieve the desired conformation of 10.6

bp/turn in the helix, exactly 500 turns are required over the length of

5300 base pairs.



Linkage number = 500 + Writhe



We can have integral values for the linkage number, and we can certainly

introduce 500, which would require no Writhe at all:



500 = 500 + 0



What is the DNA molecule was 5200 base pairs?



Apparent linkage number (Twist) = (5200 base pairs) / (10.6 base

pairs/turn)



Apparent linkage number (Twist) = 490.6



We can introduce either 490 or 491 as a linkage number, but not 490.6.

What happens if there is a linkage number of 490 in the DNA molecule?



Linkage number = Twist + Writhe

490 = 490.6 + Writhe



Writhe = -0.6



In this case, the DNA adopts a negative 0.6 supercoil (about 108° of a

right-handed supercoil) which will increase the apparent linkage number

from 490 to 490.6 (and achieve 10.6 basepairs per turn in the duplex).



How many basepairs per turn would there be in the DNA if the DNA was not

able to adopt any supercoil structure for this length of DNA with a linkage

number of 490?



Linkage number = Twist + Writhe



490 = Twist + 0



Twist = 490 turns



Twist = (5200 base pairs) / (bp/turn) = 490 turns



bp/turn = 5200 base pairs/490 turns



bp/turn = 10.61



There are slightly more than 10.6 basepairs per turn in the DNA







1998 Dr. Michael Blaber



BCH5425 Molecular Biology and Biotechnology

Spring 1998

Dr. Michael Blaber

blaber@sb.fsu.edu







Lecture 8



DNA Supercoiling, cont., topoisomerases







A small circularly closed genome

The Simian Virus 40 (SV40) genome is a circular, closed, double stranded

DNA genome. For the purposes of this discussion, it has 5300 bases. We

expect that under physiological conditions the DNA will exhibit 10.6 base

pairs per turn (i.e. one Twist = 10.6 bp/turn). In this case, with no Writhe,

the Linking number would be:



Linking number = 5300 bp/(10.6 bp/turn) + 0



Linking number = 500 turns



i.e. we would expect 500 360° turns of the DNA strands over the length

of the circular genome.



 This form (with 10.6 base pairs per turn) with no Writhe represents

the "standard", or undistorted, DNA helix.

 This is also known as the "relaxed" form of DNA, and the duplex could

physically be laid out flat on a surface because it needs no Writhe

to achieve the preferred value of 10.6 basepairs per twist:







However, when the replication of SV40 is initially completed it is

observed that there remains an open duplex region in the DNA:







The result is that there are about 475 turns of the helix within the duplex

DNA (i.e. the Linking number = 475).



 The DNA is said to be underwound.

 An open area is energetically unfavorable.

 The covalently closed molecule cannot adjust for this by increasing

the Linking number. That is, it cannot spontaneously break one or

both strands of the duplex, introduce another 25 turns into the

duplex (increase the Linking number by 25) and re-ligate the duplex.



The DNA has three choices:



1. It can adjust the number of basepairs per turn throughout the

molecule from a desired 10.6 bp/turn to 11.2 bp/turn (i.e. 5300

bp/475 turns). (NOTE: an increase in the number of basepairs per

turn will decrease the twist value; underwound DNA has a greater

number of basepairs per turn).

2. The DNA can coil up into a "supercoil" topology and maintain the

desired twist value (10.6) with the given linking number (475 in

this case).

3. The duplex can exist with a twist of 10.6 bp/turn for most of the

structure, and then have a region with zero twist (not necessarily

a melted duplex). This is quite unfavorable due to the geometry

required of bond angles.



Thus for the 5300 bp SV40 genome, with a Linking number of 475, to maintain

a value of 10.6 bp/twist, a total of 25 negative supercoils (Writhe=25)

are needed:



475 = (5300/10.6) + Writhe



-25 = Writhe



 That is, 25 negative supercoils (twenty five 180° turns of the DNA

duplex, right handed as you look down the supercoiling).



Topoisomerases



The enzymes that control DNA topology are critical to DNA replication and

transcription.



 As the replication fork opens up, the region of the duplex in front

of the fork becomes overwound - i.e. it has fewer basepairs per turn.

 The linking number has not changed, but the length of DNA which

contains all the turns is effectively shorter.

 To maintain 10.6 bp/turn in that region, the DNA will adopt positive

supercoils.



For example, during the early stages of SV40 replication, the duplex

around the origin of replication may initially melt (open up) a region

of 750 bases. Since the Linkage number (500) is unchanged, it is

effectively distributed over only:



5300 - 750 = 4550 bases.



Assuming no supercoiling has been introduced:



500 = 4550 basepairs / (X basepairs/twist) + 0



= 9.1 basepairs/twist

Thus, if no supercoiling is introduced, the DNA must adopt a conformation

of 9.01 base pairs/twist of the helix within the region ahead of the

replication fork.



 This is energetically unfavorable, and one option for the DNA is

to adopt a supercoiled configuration to achieve 10.6 bp/twist:



500 = (4550/10.6) + Writhe



70.8 = Writhe



 Thus, movement of the growing fork causes the DNA to adopt positive

supercoils.

 In this case the DNA has adopted 70.8 left handed supercoils (180°

each).

 Twist (=basepairs * [twist/basepair]) and Writhe are both real

numbers.



Type I Topoisomerase



 Type I topoisomerases cut one strand of the DNA (i.e. it "nicks"

the DNA duplex).

 The 5' phosphate of the nicked strand is covalently attached to a

tyrosine in the protein.

 The 3' end of the nick then passes once through the duplex.

 The nick is then resealed, and the linkage number is change by a

value of +1.

 This can therefore result in the removal of a single negative

supercoil.



In E. coli, type I topoisomerase can only relieve negatively supercoiled

DNA (negative supercoiling is the end result of newly replicated DNA

genome). In eukaryotes, type I topoisomerase can also relieve positively

supercoiled DNA.



The net result of E. coli topoI can be diagrammed as follows:







Type II Topoisomerases



 Type II topoisomerases actually cleave the duplex DNA in changing

the linkage number.

 Type II topoisomerases can convert a single positive supercoil into

a negative supercoil.

 Thus the linkage number is reduced by two (-2) in a single step.

 Type II topoisomerases are involved in both decatenation of

daughter chromosomes, and relieving the positive supercoiling

ahead of the replication fork.



E. coli DNA gyrase is an example of a type II topoisomerase.