MOLECULAR BIOLOGY AND BIOCHEMISTRY 694_407 by pptfiles

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									MOLECULAR BIOLOGY AND BIOCHEMISTRY 694:407 & 115:511 Handout for Lectures 16 & 17: Structures of DNA and RNA (Nov. 1 2005) Dr. Marty Nemeroff Waksman 19 (732) 445-4661 nemeroff@waksman.rutgers.edu

“A structure this pretty just had to exist.” —James D. Watson, The Double Helix, 1968

Outline I. Historical Background (Landmark Experiments in the Study of the Genetic Material) A. 1865—Mendel B. 1928—Griffith C. 1944—Avery, MacLeod, and McCarty D. 1950—Chargaff E. 1952—Hershey and Chase F. 1953—Watson and Crick (& Franklin and Wilkins) II. Nucleotides A. Structure 1. nitrogeneous bases, ribose sugar, phosphate(s) 2. N-glycosidic bonds, phosphoester bonds, phosphoanhydride bonds B. Nomenclature 1. nucleosides 2. nucleotides C. Functions 1. building blocks for DNA and RNA 2. high-energy source 3. glycolipids 4. signal transduction III. DNA A. 3-D Structure of DNA 1. phosphodiester bonds, hydrogen bonds 2. antiparallel strands 3. major and minor grooves B. Secondary structure 1. direct repeats and mirror repeats 2. inverted repeats C. Function(s) 1. storage of information (encode genes, regulatory sequences, processing signals) D. Properties 1. very stable 2. detection 3. denaturation 4. precipitation

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IV. RNA A. Secondary and 3-D structure 1. RNA forms intra-strand hydrogen bonds 2. 3-D structure affected by secondary structure B. Functions 1. carries genetic information 2. structural molecules, splicing, translation, ribozymes C. Properties 1. very unstable 2. degraded by alkali treatment 3. denaturation V. What You Need to Know for Exam #3 Reading list A. Biochemistry, Garrett and Grisham, 3rd Edition 1. Chapter 10—Nucleotides and Nucleic Acids 2. Chapter 11—Structure of Nucleic Acids B. Molecular Biology of the Gene, Watson et al, 5th Edition 1. Chapter 6—The Structures of DNA and RNA

I. Landmark Experiments on the Genetic Material
A. 1865—Gregor Mendel noted that physical traits are inherited as discrete units: “Genes” B. 1928—Frederick Griffith injected bacteria into mice”The Transforming Principle”

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C. 1944—Avery, MacLeod, and McCartyWhat is the Transforming Principle? Treatment proteases lipases DNase RNase Transformation?

D. 1951 Erwin Chargaff—DNA molecules Have Distinctive Base Compositions Base composition of DNA (mol %) and ratios of base pairs: Chargaff’s Rules Source
Escherichia coli Micobacterium tuberculosis Yeast Ox Pig Humans

A
26.0 15.1 31.7 29.0 29.8 30.4

G
24.9 34.9 18.3 21.2 20.7 19.9

C
25.2 35.4 17.4 21.2 20.7 19.9

T
23.9 14.6 32.6 28.7 29.1 30.1

A/T
1.088 1.034 0.972 1.010 1.024 1.010

G/C
0.988 0.986 1.052 1.000 1.000 1.000

G+C
50.1 70.3 35.7 42.4 41.4 39.8

Purine/ Pyrimidine
1.037 1.00 1.00 1.006 1.014 1.006

1. The base composition of DNA varies from one species to another. 2. DNA specimens from different tissues of the same species have the same base composition. 3. The base composition doesn’t change with age, nutritional state, or changing environment. 4. In all cellular DNAs, regardless of species, the number of adenosine residues is the same as the number of thymidine residues and #guanosine=#cytosine residues.

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E. 1952 Hershey and Chase—What is the genetic material of bacteriophage T4?

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F. 1953—Watson and Crick (& Wilkins and Franklin)

Crystalline Occurs at 75% rel. humidity Contains 30% water

Paracrystalline At >75% rel. humidity Contains >30% water

Structure A of DNA (Maurice Wilkins)

Structure B of DNA (Rosalind Franklin)

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II. Nucleotides

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Base Purines Adenine

Nucleoside

Nucleotide

Nucleic Acid

Adenylate (Deoxyadenylate) Guanylate (Deoxyguanylate)

RNA (DNA)

Guanine

Pyrimidines Cytosine Cytidylate (Deoxycytidylate) Thymine (Thymidylate) or (Deoxythmidylate)

Uracil

Uridylate

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A Nucleotide

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Conformation of bases when attached to ribose

Nucleoside monophosphates can converted to nucleoside di- and triphosphates

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Roles of Nucleotides
Deoxyribonucleotides —dNTPs are the building blocks for making DNA Ribonucleotides —NTPs are the building blocks for making RNA —ATP drives many reactions —GTP used for protein synthesis (initiation and elongation steps) —CTP use for lipid synthesis (CDP donates the phosphate group in glycerophospholipids) —UTP used for carbohydrate metabolism (UDP-glucose + fructosesucrose) —cyclic nucleotides (cAMP and cGMP) are signal molecules (epinephrine hormone pathway)

Unusual bases of RNA

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The Stucture of DNA

6Å

3.4Å 34Å

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The two DNA strands are held together by hydrogen bonds

What are hydrogen bonds?

The DNA strands run antiparallel

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The double helix has alternating major and minor grooves

How is DNA synthesized?

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B-DNA

36 bp

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Nucleic acids absorb UV light
You can quantify the concentration of your nucleic acid by measuring the UV absorbance UV light absorbance increases as DNA goes from double stranded to single stranded “Hyperchromic Shift”

Melting temperature Tm increases with G:C content

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Cot Curve Analysis of DNAs
The rate of reassociation of denatured DNA is inversely proportional to genome complexity.

C0 = Conc ssDNA at t=0

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Another way to detect DNA is by ethidium bromide staining

Renaturation Kinetics = DNA Complexity

DNA can be concentrated by precipitation

Requires salt Requires ethanol

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Alkali affects DNA and RNA differently
Denatures DNA Hydrolyzes RNA

RNAs form intrastrand base pairing

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RNA plays many different roles in the cell
DNA -stores the genetic material RNA -messenger RNAs (mRNAs) to make proteins -transfer RNAs (tRNAs) to carry amino acids to the ribosome -ribosomal RNAs (rRNAs) structural components of ribosomes -small nuclear RNAs (snRNAs) for pre-mRNA splicing -catalytic RNAs (Ribozymes) can catalyze A) RNA cleavage reactions (e.g., RNaseP = cleaves the 5’ ends of tRNAs) B) transesterification reactions (e.g., group I and group II introns = self-splicing C) peptide bond formation (e.g., the 23S rRNA in the 50S ribosomal subunit has the “peptidyl transferase” activity Examples of Catalytic RNAs

A) RNaseP cleavage at tRNA 5’ ends)

B) Group II intron (self-splicing)

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C) Peptidyl transferase of 23S rRNA How can some RNAs can by catalytic?

V. What you need to know for Exam #3:
I. Historical Background 1. Conclusions (and significance) of landmark experiments in molecular biology II. Nucleotides 1. Be able to identify the five Watson & Crick nitrogenous bases 2. The composition of nucleosides, and nucleotides (in what way are nucleosides different from nucleotides, etc.?) 3. Nomenclature of nucleosides and nucleotides (if you add a sugar and a 5’ phosphate to cytosine, what is it now called?) 4. The covalent bonds that link the components of nucleotides 5. The structural differences are between ribonucleosides and deoxyribonucleosides III. & IV DNA and RNA 1. Chargaff’s rules 2. Structure of DNA: double-stranded, antiparallel strands, hydrogen bonds 3. Bonds that hold DNA together 4. Dimensions of the double helix (pitch, width, bp/turn) 5. Differences between A, B, and Z DNA 6. Can you tell if a helix is right-handed and left-handed? 7. How to break hydrogen bonds in DNA 8. How to interpret cot curves and what cot curves tell you about DNAs from different sources 8. What does alkali do to RNA? 9. Why is DNA synthesis thermodynamically favorable? 10. Why does RNA not have a regular 3-D structure like DNA? 11. Why is only RNA capable of being catalytic? 12. What ways are there of detecting DNA or RNA? 13. Which is more stable: DNA or RNA?

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