Chapter 10 Nucleotides and Nucleic Acids

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Chapter 10 Nucleotides and Nucleic Acids Powered By Docstoc
					     Reginald H. Garrett
     Charles M. Grisham




         Chapter 10
Nucleotides and Nucleic Acids
 Outline

• What are the structure and chemistry of
  nitrogenous bases ?
• What are nucleosides ?
• What are the structure and chemistry of
  nucleotides ?
• What are nucleic acids ?
• What are the different classes of nucleic Acids ?
• Are nucleic acids susceptible to hydrolysis ?
Information Transfer in Cells

      Central Dogma of Molecular Biology

• Information encoded in a DNA molecule is
  transcribed via synthesis of an RNA molecule.
• The sequence of the RNA molecule is "read"
  and is translated into the sequence of amino
  acids in a protein.
• See Figure 10.1.
Information Transfer in Cells

Figure 10.1
The fundamental
process of
information transfer
in cells.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
Figure 10.2(a) The pyrimidine ring system; by convention,
atoms are numbered as indicated. N1 is attached to ribose.




 (b) The purine ring system; atoms numbered as shown.
 N9 is attached to ribose.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
               Know these structures




  Figure 10.3 The common pyrimidine bases –
  cytosine, uracil, and thymine – in the tautomeric forms
  predominant at pH 7.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?
               Know these structures




 Figure 10.4 The common purine bases – adenine and
 guanine – in the tautomeric forms predominant at pH 7.
10.1 What Are the Structure and
Chemistry of Nitrogenous Bases?


Figure 10.5
Other naturally occurring purine
derivatives – hypoxanthine, xanthine,
and uric acid.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature
  • The aromaticity and electron-rich nature of
    pyrimidines and purines enable them to undergo
    keto-enol tautomerism.
  • The keto tautomers of uracil, thymine, and
    guanine predominate at pH 7.
  • By contrast, the enol form of cytosine
    predominates at pH 7.
  • Protonation states of the nitrogens determines
    whether they can serve as H-bond donors or
    acceptors.
  • Aromaticity also accounts for strong absorption of
    UV light at 260 nm. (Proteins absorb at 280 nm.)
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature




     Figure 10.6 The keto-enol tautomerism of uracil.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature




   Figure 10.7 The tautomerization of the purine guanine.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature




    Figure 10.8 The UV absorption spectra of the common
    ribonucleotides.
The Properties of Pyrimidines and Purines Can
Be Traced to Their Electron-Rich Nature




     Figure 10.8 The UV absorption spectra of the common
     ribonucleotides.
10.2 What Are Nucleosides?

                Structures to Know
 • Nucleosides are formed when a base is linked to
   a sugar via a beta glycosidic bond.
 • The sugars are pentoses.
 • D-ribose (in RNA).
 • 2-deoxy-D-ribose (in DNA).
 • The difference - 2'-OH vs 2'-H. Primes are used
   in nucleosides and nucleotides but not sugars
   alone.
 • This difference affects secondary structure and
   stability.
10.2 What Are Nucleosides?




 Figure 10.9 The linear   Figure 10.9 The linear
 (Fischer) and cyclic     (Fischer) and cyclic
 (furanose) forms of      (furanose) forms of
 ribose.                  deoxyribose.
10.2 What Are Nucleosides?


 • The base is linked to the sugar via a beta
   glycosidic bond.
 • The carbon of the glycosidic bond is anomeric.
 • Named by adding -idine to the root name of a
   pyrimidine or -osine to the root name of a purine.
   (Uracil  uridine and adenine  adenosine)
 • Conformation can be syn or anti.
 • Sugars make nucleosides more water-soluble
   than free bases.
10.2 What Are Nucleosides?




       Figure 10.10 The common ribonucleosides.
10.3 What Is the Structure and Chemistry
of Nucleotides?


     Nucleotides are nucleoside phosphates
  • Know the nomenclature.
  • "Nucleotide phosphate" is redundant!
  • Most nucleotides are ribonucleotides.
  • Nucleotides are polyprotic acids due to the
  phosphates.
10.3 What Is the Structure and Chemistry
of Nucleotides?




  Figure 10.11 Structures of the four common ribonucleotides –
  AMP, GMP, CMP, and UMP. Also shown: 3’-AMP.
10.3 What Is the Structure and Chemistry
of Nucleotides?




Figure 10.13 Formation of ADP from AMP by the addition of a
phosphate group forming a phosphoric anhydride linkage.
Note that the reaction is a dehydration synthesis.
10.3 What Is the Structure and Chemistry
of Nucleotides?




Figure 10.13 Formation of ATP from ADP by the addition of a
phosphate group forming a phosphoric anhydride linkage.
This is also a dehydration process.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy

  Nucleoside 5’-triphosphates are indispensable
    agents in metabolism because their phosphoric
    anhydride bonds are a source of chemical
    energy.
  Functions:
  • 1. ATP is central to energy metabolism: (see the
    following slides).
  • 2. Nucleotides serve as signal molecules and
    regulators:      c-AMP and c-GMP.
  • 3. NTPs are substrates for DNA and RNA
    synthesis. The bases serve as recognition units.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
Functions continued:
• 4. Nucleotides are high energy carrier molecules:
   • GTP is involved in protein synthesis (translation).
      • Initiation, elongation and termination steps.
      • And ATP in activation: Aminoacyl-AMP.
   • CTP is involved in lipid synthesis.
      • CDP-diacylglycerol, etc.
   • UTP is involved in carbohydrate metabolism.
      • UDP-glucose, etc.
• 5. Nucleotides are redox cofactors:
   • NAD+, NADP+, FMN, FAD, Coenzyme A.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy
Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidyl
group transfer, the major biochemical reactions of nucleotides.




      Phosphoryl group transfer is shown here.




     Pyrophosphoryl group transfer is shown here.
Nucleoside 5'-Triphosphates Are Carriers
of Chemical Energy




      Nucleotidyl group transfer is shown here.
10.3 What Is the Structure and Chemistry
of Nucleotides?




 Figure 10.12 The cyclic   Figure 10.12 The cyclic
 nucleotide cAMP.          nucleotide cGMP
10.4 What Are Nucleic Acids?


 • Nucleic acids are linear polymers of nucleotides
   linked 3' to 5' by phosphodiester bonds.
 • Two types:
    • Ribonucleic acid and
    • Deoxyribonucleic acid.
 • Know the shorthand notations.
 • Sequence is always read 5' to 3', left to right.
 • In terms of genetic information, this corresponds
   to "N-terminal to C-terminal“ in proteins.
10.4 What Are Nucleic Acids?


Figure 10.15 RNA
3',5'-phosphodiester bridges
link nucleotides together to
form polynucleotide chains.
The 5'-ends of the chains
are at the top; the 3'-ends
are at the bottom.
10.4 What Are Nucleic Acids?




Figure 10.15 DNA:
3’,5’-phosphodiester bridges
link nucleotides together to
form polynucleotide chains.
The 5’-ends of the chains
are at the top; the 3’-ends
are at the bottom.
10.4 What Are Nucleic Acids?

  Shorthand notation for DNA.
                 A       G           T       A       C
              1'

                             a
              3'

          P          P           P       P       P       OH
              5'                 b

 The bases are at the top.
 The vertical line is the sugar numbered top to bottom.
 The 5' end is to the left and the 3' end to the right.
 a and b are cleavage sites for nucleases.
 Linkage is a phosphodiester bond, each P has a (-) charge.
10.5 What Are the Different Classes of
Nucleic Acids?
 • DNA - one type, one purpose (three forms).
 • RNA - Several types, several purposes:
   • ribosomal RNA - the basis of structure and
     function of ribosomes (largest amount).
   • messenger RNA - carries the message for
     protein synthesis (fewest and unique).
   • transfer RNA - carries the amino acids for
     protein synthesis (smallest molecules).
   • Others:
       • Small nuclear RNA.
       • Small non-coding RNAs.
       • Viral
10.5 What Are the Different Classes of
Nucleic Acids?

Figure 10.16
The antiparallel nature of
the DNA double helix.
The DNA Double Helix
  The double helix is stabilized by hydrogen
     bonds and hydrophobic interactions
• "Base pairs" arise from hydrogen bonds.
• Erwin Chargaff had the pairing data, but didn't
  understand its implications.
• Rosalind Franklin's X-ray fiber diffraction data
  was crucial.
• Francis Crick showed that it was a helix.
• James Watson figured out the H bonds.
• The hydrophobic effect from stacking of
  aromatic bases is also important.
Chargaff’s Data Held the Clue to Base
Pairing
The Base Pairs Postulated by Watson




  Figure 10.17 The Watson-Crick base pairing in A:T.
         Practice drawing this structure
The Base Pairs Postulated by Watson




 Figure 10.17 The Watson-Crick base pairing in G:C.
              Practice drawing this structure.
The Structure of DNA

           An antiparallel double helix
 • Has a diameter of 2 nm.
 • Has a length of 1.6 million nm in E. coli.
 • Compact and folded (E. coli cell is only 2000
   nm long).
 • Eukaryotic DNA is wrapped around histone
   proteins to form nucleosomes.
 • Base pairs: A-T, G-C.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein

          Transcription product of DNA

  • In prokaryotes, a single mRNA contains the
    information for synthesis of many proteins
  • In eukaryotes, a single mRNA codes for just
    one protein, but structure is composed of
    introns and exons
  • See following slides.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein
   Figure 10.20 Transcription and translation of mRNA
   molecules in prokaryotic versus eukaryotic cells.




    In prokaryotes, a single mRNA molecule may contain the
    information for the synthesis of several polypeptide
    chains within its nucleotide sequence.
Messenger RNA Carries the Sequence
Information for Synthesis of a Protein




  In eukaryotics, mRNAs encode only one polypeptide but
  are more complex.
Eukaryotic mRNA

• In eucaryotes, DNA is transcribed to produce
  heterogeneous nuclear RNA:
   • mixed introns and exons with poly A.
   • intron - intervening sequence.
   • exon - coding sequence.
   • poly A tail – stability ?
• Splicing produces final mRNA without introns.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
 • Ribosomes are about 2/3 RNA, 1/3 protein.
 • rRNA serves as a scaffold for ribosomal proteins.
 • The different species of rRNA are referred to
   according to their sedimentation coefficients.
 • rRNAs typically contain certain modified
   nucleotides, including pseudouridine and
   ribothymidylic acid.
 • The role of ribosomes in biosynthesis of proteins
   is treated in detail in Chapter 30.
 • Briefly: the genetic information in the nucleotide
   sequence of mRNA is translated into the amino
   acid sequence of a polypeptide chain by the
   ribosomes.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes
Figure 10.21
Ribosomal RNA has a
complex secondary structure
due to many intrastrand H
bonds.
The gray line here traces a
polynucleotide chain
consisting of more than 1000
nucleotides.
Aligned regions represent H-
bonded complementary base
sequences.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes




Figure 10.22 The organization and composition of ribosomes.
Transfer RNAs Carry Amino Acids to
Ribosomes for Use in Protein Synthesis

  • tRNAs are small polynucleotide chains.
     • 73 to 94 residues each, ~10% minor bases.
  • Several bases are usually methylated.
  • Each a.a. has at least one unique tRNA which
    carries the a.a. to the ribosome.
  • The 3'-terminus carries the amino acid and the
    3'-terminal sequence is always CCA-a.a.
  • A tRNA with an amino acid attached is called
    an Aminoacyl tRNA. These molecules are the
    substrates for protein synthesis.
Ribosomal RNA Provides the Structural
and Functional Foundation for Ribosomes

Figure 10-23
Some unusual bases
in DNA.
Transfer RNAs Carry Amino Acids to
Ribosomes for Use in Protein Synthesis

Figure 10.24
Transfer RNA also has a
complex secondary
structure due to many
intrastrand hydrogen
bonds.
The black lines represent
base-paired nucleotides
in the sequence.
The Chemical Differences Between DNA
and RNA Have Biological Significance
 • Two fundamental chemical differences distinguish
   DNA from RNA:
   • DNA contains 2-deoxyribose instead of ribose.
   • DNA contains thymine instead of uracil.

         Why does DNA contain thymine ?
 • Cytosine spontaneously deaminates to form uracil.
 • Repair enzymes recognize these "mutations" and
   replace these Us with Cs.
 • But how would the repair enzymes distinguish
   natural U from mutant U.
 • Nature solves this dilemma by using thymine (5-
   methyl-U) in place of uracil.
The Chemical Differences Between DNA
and RNA Have Biological Significance




   Figure 10.25 Deamination    Figure 10.26 The 5-
   of cytosine forms uracil.   methyl group on
                               thymine labels it as a
                               special kind of uracil.
DNA & RNA Differences?

    Why is DNA 2'-deoxy and RNA is not?
• Vicinal -OH groups (2' and 3') in RNA make it
  more susceptible to hydrolysis
• DNA, lacking 2'-OH is more stable
• This makes sense - the genetic material must
  be more stable
• RNA is designed to be used and then broken
  down
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?


    •   RNA is resistant to dilute acid.
    •   DNA is depurinated by dilute acid.
    •   DNA is not susceptible to base.
    •   RNA is hydrolyzed by dilute base.
    •   See Figure 10.29 for mechanism.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




  Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack
  by OH- on the P atom leads to 5'-phosphoester cleavage.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




Figure 10.27 Alkaline hydrolysis of RNA. Random hydrolysis
of the cyclic phosphodiester intermediate gives a mixture of 2'-
and 3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




Figure 10.27 Alkaline hydrolysis of RNA. The mixture of 2'- and
3'-nucleoside monophosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




Figure 10.28 Cleavage in polynucleotide chains. Cleavage
on the a-side leaves the phosphate attached to the 5'-
position of the adjacent nucleotide. b-side hydrolysis yields
3'-phosphate products.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




  Figure 10.28 Cleavage in polynucleotide chains.
  Cleavage on the a-side leaves the phosphate attached to
  the 5'-position of the adjacent nucleotide.

  Nucleic acids are cleaved by nucleases which may be “a”
  or “b” types. Some are exonucleases and some are
  endonucleases.
10.6 Are Nucleic Acids Susceptible to
Hydrolysis?




    Figure 10.28 Cleavage in polynucleotide chains.
    b-side hydrolysis yields 3'-phosphate products.
  Restriction Enzymes
• Bacteria "restrict" the possibility of attack from
  foreign DNA by means of "restriction enzymes".
      Methylation protects DNA of the bacteria.
• Three known types of restriction enzymes:
   • Type I – has endonuclease and methylase
     activity and cleaves about 1000 bp from an
     unmethylated recognition sequence.
   • Type II – cleaves dsDNA only in an
     unmethylated recognition sequence.
   • Type III – has endonuclease and methylase
     activity and cleaves about 25 bp from an
     unmethylated recognition sequence.
Restriction Enzymes

• Type II restriction enzymes cleave DNA
  chains at selected sites (most useful in lab).
• Enzymes may recognize 4, 6 or more bases
  in selecting sites for cleavage.
• An enzyme that recognizes a 6-base
  sequence is a "six-cutter“.
• Type II enzymes are specific and do not
  require ATP.
• Types I and III are less specific. Type I
  requires ATP and Type III is ???.
Type II Restriction Enzymes

• Recognition sites in dsDNA for Type II have a
  2-fold axis of symmetry (palindromic).
• Cleavage can produce staggered or "sticky"
  ends or "blunt” ends depending on the
  enzyme.
• Names use 3-letter italicized code:
   • 1st letter - genus; 2nd,3rd – species.
• Following letter denotes strain.
• Roman numeral = number of enzyme found.
• EcoRI is the first restriction enzyme found in
  the R strain of E. coli.
Cleavage Sequences of Restriction
Endonucleases
Cleavage Sequences of Restriction
Endonucleases
Cleavage Sequences of Restriction
Endonucleases
Restriction Mapping of DNA

Figure 10.29 Restriction mapping analysis.
Restriction Mapping of DNA

 Figure 10.29 Restriction mapping analysis.
      End Chapter 10
Nucleotides and Nucleic Acids

				
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