Lehninger Principles of Biochemistry

1
Lehninger Principles of Biochemistry Fourth
Edition
Chapter 8 Nucleotides and Nucleic Acids
2
(Some) Chapter 8 Objectives

• To understand the structure of nucleic acids and
  their monomeric composites, nucleotides
• To become familiar with the forces that
  contribute to the formation of DNA and RNA
• To discuss the physical and chemical properties
  of nucleic acids and modern DNA synthesis

3
Nucleotides in Biochemistry

• Monomeric units of nucleic acids
• NTPs are the end products of most
  energy-producing pathways (High energy
  compounds)
• Most metabolic pathways are regulated by levels
  of AMP, ADP, and ATP some hormonal signals are
  regulated by cAMP and/or cGMP
• Adenine nucleotides are components of many
  coenzymes (e.g., FAD, NAD, NADP, CoASH, etc.)

4
Nucleotide Structure

• Nitrogen-containing base pentose phosphate
  nucleotide
• Nucleotide (phosphate) nucleoside

HO
HO
5
(No Transcript)
6
Bases
Cytosine, Thymine (Uracil)
Adenine, Guanine
7
(No Transcript)
8
(No Transcript)
9
(No Transcript)
10
NOTE There are several naturally occurring
minor bases, modified by addition of heteroatoms
(e.g., S), methylation, and different sites of
sugar attachment and phosphorylation.
11
(No Transcript)
12
DNA and RNA are hydrolyzed slowly in solution,
but only RNA undergoes base-catalyzed rapid
hydrolysis. WHY?
13
(No Transcript)
14

• Tautermization occurs with changes in pH
• Highly conjugated ring systems absorb strongly in
  the UV
• Planar bases stack (?-stacking)

15
The 3-D Structure of DNASome basics

• Chargoffs rules Base ratios in DNA Known since
  1940 that in DNA, GC and AT (AG CT)
• Tautomeric forms that predominate were solved via
  NMR, X-ray diffraction
• Rosalind Franklins X-ray diffraction photograph
  revealed helical structure

16
The DNA Story

• 1869 Friedrich Miescher isolates nucleic acids
  from dried blood
• 1928 F. Griffith finds heat-killed virulent
  bacteria transformed live non-virulent bacteria
  
• 1944 Avery, MacLeod, and Maclyn report the
  transforming principle to be DNA

17
Griffiths experiment
Pneumococcus (Diplococcus pneumoniae) Wild Type
(S) Polysaccharide coat (smooth) contains O
antigens required for recognition of target
cell Mutant line (R) Lack polysaccharide coat
(rough) non-pathogenic

• Avery, MacLeod, and Maclyn conclude that DNA
  transformed non-pathogenic bacteria to virulent
  form.
• How?
• Had physical and chemical properties of DNA,
• Highly purified prep contained no protein (was
  not affected by proteases), and
• DNAse completely inactivated process

18
The DNA Story

• 1869 Friedrich Miescher isolates nucleic acids
  from dried blood
• 1928 F. Griffith finds heat-killed virulent
  bacteria transformed live non-virulent bacteria
  
• 1944 Avery, MacLeod, and Maclyn report the
  transforming principle to be DNA
• 1952 A. Hershey and M. Chase provide additional
  evidence that DNA is the carrier of genetic
  material

19
The Hershey-Chase Experiment
20
The DNA Story

• 1869 Friedrich Miescher isolates nucleic acids
  from dried blood
• 1928 F. Griffith finds heat-killed virulent
  bacteria transformed live non-virulent bacteria
  
• 1944 Avery, MacLeod, and Maclyn report the
  transforming principle to be DNA
• 1952 A. Hershey and M. Chase provide additional
  evidence that DNA is the carrier of genetic
  material
• 1950s X-ray diffraction patters, Chargoffs
  rules, structural properties lead Watson and
  Crick to solve the structure (1953)

21
(No Transcript)
22
(No Transcript)
23
(No Transcript)
24
Watson-Crick BP
25
(No Transcript)
26
Some common local DNA motifs
27
Ends of linear chromosomes (telomeres produced
through enzymatic reaction by telomerase) are
rich in G (poly-dG) and thus form tetraplex
structure. Telomeres naturally shorten as part
of the aging process. Thus, the loss of
telomerase function is a basis for aging. Ovarian
cancer cells display high telomerase
activity. Telomerase is inhibited by tetraplex
DNA, so agents that stabilize this structure are
potential candidates as anti-tumor agents.
28
mRNA
Prokaryotic mRNA
Single stranded, right-handed RNA single helix is
the product of transcription.
29

• RNA structure can be complex
• Base pairing can form duplex (usually A form)
• tRNA and rRNA are complex forms combining the
  nucleic acid component with a protein sequence
• Some enzymes employ sequences of RNA (ribozymes)

30
Physical and Chemical Properties of Nucleic Acids

• Heat denatures (melts) double-stranded nucleic
  acids
• Structure is maintained NOT by hydrogen bonds but
  by the hydrophobic effect, although details are
  poorly understood Base stacking

31
Heat (and changes in pH/ionic strength) disrupts
hydrogen bonds and van der Waals and stacking
interactions, causing denaturation. (Covalent
bonds remain intact.) If complete separation does
not occur, the duplex can re-anneal
(spontaneously) under proper conditions. If
strands completely separate, the process is
slower and step-wise.
32
The Hypo(er)chromic Effect

• Because of high degree of conjugation, pi
  electrons cause high molar extinction
  coefficients
• Polynucleotides absorb LESS than monomeric
  nucleotides, but at the same wavelength (? 260
  nm) (Hypochromic)
• Also, single stranded DNA absorbs MORE than
  double stranded
• This can be used to monitor the kinetics of DNA
  melting

33
QUESTIONS 1. What does the shape of the melting
curve say about the process? 2. a. Why does GC
to AT ratio affect Tm? b. Consider the
following empirical relationship Tm 41.1XGC
16.6 log Na 81.5
34
Some important nucleic acid reactions

• Degree of hybridization between complimentary DNA
  strands from different organisms is used to
  determine evolutionary relations
• Spontaneous deamination
• Cytosine minus amino group uracil
• What are the implications of this for DNA?
• Depurination
• Dimerization

35
(No Transcript)
36
Some important nucleic acid reactions

• Degree of hybridization between complimentary DNA
  strands from different organisms is used to
  determine evolutionary relations
• Spontaneous deamination
• Cytosine minus amino group uracil
• What are the implications of this for DNA?
• Depurination
• Dimerization (UV light)
• Methylation (methyl donor is S-Adenosylmethionine
  SAM)

37
DNA Sequencing The Sanger Method
HOMEWORK Problem 11 (p. 304)
38
DNA ElectrophoresisSouthern Blotting
39
(No Transcript)
40
(No Transcript)