1. Nucleic Acids: Cell Overview and Core Topics

2. Outline
Cellular Overview
Anatomy of the Nucleic Acids
Building blocks
Structure (DNA, RNA)
Looking at the Central Dogma
DNA Replication
RNA Transcription
Protein Synthesis

3. DNA and RNA in the Cell
Cellular Overview

4. Classes of Nucleic Acids: DNA
DNA is usually found in the nucleus
Small amounts are also found in:
mitochondria of eukaryotes
chloroplasts of plants
Packing of DNA:
2-3 meters long
histones
genome = complete collection of hereditary information of an organism

5. Classes of Nucleic Acids: RNA
FOUR TYPES OF RNA
• mRNA - Messenger RNA • tRNA - Transfer RNA • rRNA - Ribosomal RNA • snRNA - Small nuclear RNA

6. Anatomy of Nucleic Acids
THE BUILDING BLOCKS
Anatomy of Nucleic Acids

7. Nucleic acids are linear polymers.
Each monomer nucleotide consists of:
1. a sugar
2. a phosphate
3. a nitrogenous base

8. Nitrogenous Bases

9. Why ? Nitrogenous Bases DNA (deoxyribonucleic acid):
adenine (A) guanine (G)
cytosine (C) thymine (T)
Why ?
RNA (ribonucleic acid):
adenine (A) guanine (G)
cytosine (C) uracil (U)

10. Properties of purines and pyrimidines:
keto – enol tautomerism
strong UV absorbance

11. Pentose Sugars of Nucleic Acids
This difference in structure affects secondary structure and stability.
Which is more stable?

12. Nucleosides
linkage of a base and a sugar.

13. Nucleotides - nucleoside + phosphate - monomers of nucleic acids
- NA are formed by 3’-to-5’ phosphodiester linkages

14. Shorthand notation: sequence is read from 5’ to 3’
corresponds to the N to C terminal of proteins

15. Nucleic Acids: Structure
DNA
Nucleic Acids: Structure

16. Primary Structure
nucleotide sequences

17. Secondary Structure DNA Double Helix Features:
Maurice Wilkins and Rosalind Franklin
James Watson and Francis Crick
Features:
two helical polynucleotides coiled around an axis
chains run in opposite directions
sugar-phosphate backbone on the outside, bases on the inside
bases nearly perpendicular to the axis
repeats every 34 Å
10 bases per turn of the helix
diameter of the helix is 20 Å

19. Double helix stabilized by hydrogen bonds.
Which is more stable?

20. Axial view of DNA

21. A and B forms are both right-handed double helix.
A-DNA has different characteristics from the more common B-DNA.

22. Z-DNA
left-handed
backbone phosphates zigzag

23. Comparison Between A, B, and Z DNA:
A-DNA: right-handed, short and broad, 11 bp per turn
B-DNA: right-handed, longer, thinner, 10 bp per turn
Z-DNA: left-handed, longest, thinnest, 12 bp per turn

24. Major and minor grooves are lined with sequence-specific H-bonding.

25. Tertiary Structure
Supercoiling
supercoiled DNA
relaxed DNA

26. Consequences of double helical structure:
1. Facilitates accurate hereditary information transmission
Reversible melting
melting: dissociation of the double helix
melting temperature (Tm)
hypochromism
annealing

27. Structure of Single-stranded DNA
Stem Loop

28. Nucleic Acids: Structure
RNA
Nucleic Acids: Structure

29. Secondary Structure
transfer RNA (tRNA) : Brings amino acids to ribosomes during translation

30. Transfer RNA
Extensive H-bonding creates four double helical domains, three capped by loops, one by a stem
Only one tRNA structure (alone) is known
Many non-canonical base pairs found in tRNA

31. Ribosomes synthesize proteins
ribosomal RNA (rRNA) : Makes up the ribosomes, together with ribosomal proteins.
Ribosomes synthesize proteins
All ribosomes contain large and small subunits
rRNA molecules make up about 2/3 of ribosome
Secondary structure features seem to be conserved, whereas sequence is not
There must be common designs and functions that must be conserved

32. messenger RNA (mRNA) : Encodes amino acid sequence of a polypeptide

33. small nuclear RNA (snRNA) :With proteins, forms complexes that are used in RNA processing in eukaryotes. (Not found in prokaryotes.)

34. DNA Replication, Recombination, and Repair
Central Dogma

35. Central Dogma

36. strand separation followed by copying of each strand
DNA Replication – process of producing identical copies of original DNA
strand separation followed by copying of each strand
fixed by base-pairing rules

37. DNA replication is bidirectional.
involves two replication forks that move in opposite direction

47. DNA replication requires unwinding of the DNA helix.
expose single-stranded templates
DNA gyrase – acts to overcome torsional stress imposed upon unwinding
helicases – catalyze unwinding of double helix
disrupts H-bonding of the two strands
SSB (single-stranded DNA-binding proteins) – binds to the unwound strands, preventing re-annealing

48. Primer
RNA primes the synthesis of DNA.
Primase synthesizes short RNA.

49. DNA replication is semidiscontinuous
DNA polymerase synthesizes the new DNA strand only in a 5’3’ direction. Dilemma: how is 5’  3’ copied?
The leading strand copies continuously
The lagging strand copies in segments called Okazaki fragments (about nucleotides at a time) which will then be joined by DNA ligase

52. DNA Polymerase All DNA Polymerases share the following:
= enzymes that replicate DNA
All DNA Polymerases share the following:
Incoming base selected in the active site (base-complementarity)
Chain growth 5’  3’ direction (antiparallel to template)
Cannot initiate DNA synthesis de novo (requires primer)
First DNA Polymerase discovered – E.coli DNA Polymerase I (by Arthur Kornberg and colleagues)
Arthur Kornberg
1959 Nobel Prize in Physiology and Medicine
Roger D. Kornberg
2006 Nobel Prize in Chemistry

53. 3’  5’ exonuclease activity
- removes incorrect nucleotides from the 3’-end of the growing chain (proofreader and editor)
- polymerase cannot elongate an improperly base-paired terminus
proofreading mechanisms
Klenow fragment – removes mismatched nucleotides from the 3’’ end of DNA (exonuclease activity)
detection of incorrect base
incorrect pairing with the template (weak H-bonding)
unable to interact with the minor groove (enzyme stalls)

54. DNA ligase seals breaks in the double stranded DNA
= seals the nicks between Okazaki fragments
DNA ligase seals breaks in the double stranded DNA
DNA ligases use an energy source (ATP in eukaryotes and archaea, NAD+ in bacteria) to form a phosphodiester bond between the 3’ hydroxyl group at the end of one DNA chain and 5’-phosphate group at the end of the other.

62. Like E. coli, but more complex
Eukaryotic DNA Replication
Like E. coli, but more complex
Human cell: 6 billion base pairs of DNA to copy
Multiple origins of replication: 1 per base pairs
E.coli chromosome
Human
E.coli circular chromosome;
Human linear

63. DNA Recombination = natural process of genetic rearrangement
recombinases
Holliday junction – crosslike structure

64. DNA replication error rate: 3 bp during copying of 6 billion bp
Mutations
Substitution of base pair
transition
transversion
Deletion of base pair/s
Insertion/Addition of base pair/s
Macrolesions: Mutations involving changes in large portions of the genome
DNA replication error rate: 3 bp during copying of 6 billion bp

65. Agents of Mutations Physical Agents UV Light Ionizing Radiation
Chemical Agents
Some chemical agents can be classified further into
Alkylating
Intercalating
Deaminating
Viral

66. UV Light Causes Pyrimidine Dimerization
Replication and gene expression are blocked

68. Chemical mutagens
5-bromouracil and 2-aminopurine can be incorporated into DNA

69. Deaminating agents Ex: Nitrous acid (HNO2)
Converts adenine to hypoxanthine, cytosine to uracil, and guanine to xanthine
Causes A-T to G-C transitions

70. Alkylating agents

71. Intercalating agents

73. Acridines Intercalate in DNA, leading to insertion or deletion
The reading frame during translation is changed

74. DNA Repair Direct repair Base excision repair
Photolyase cleave pyrimidine dimers
Base excision repair
E. coli enzyme AlkA removes modified bases such as 3- methyladenine (glycosylase activity is present)
Nucleotide excision repair
Excision of pyrimidine dimers (need different enzymes for detection, excision, and repair synthesis)

76. RNA Transcription
Central Dogma

77. Process of Transcription has four stages:
Binding of RNA polymerase at promoter sites
Initiation of polymerization
Chain elongation
Chain termination

78. Transcription (RNA Synthesis)
RNA Polymerases
Template (DNA)
Activated precursors (NTP)
Divalent metal ion (Mg2+ or Mn2+)
Mechanism is similar to DNA Synthesis

79. Limitations of RNAP II:
Reece R. Analysis of Genes and Genomes p47.
Limitations of RNAP II:
It can’t recognize its target promoter and gene. (BLIND)
It is unable to regulate mRNA production in response to developmental and environmental signals. (INSENSITIVE)

80. Start of Transcription
Promoter Sites
Where RNA Polymerase can indirectly bind

81. Preinitiation Complex (PIC)
TATA box – a DNA sequence (5’—TATAA—3’) found in the promoter region of most eukaryotic genes.
TFIID
binds to TATA; promotes TFIIB binding
TFIIA
stabilizes TBP binding
TFIIB
promotes TFIIF-pol II binding
TFIIF
targets pol II to promoter
TFIIE
stimulates TFIIH kinase and ATPase actiivities
TFII H
helicase, ATPase, CTD kinase activities
Abeles F, et al. Biochemistry p391.
Transcription Factors (TF):
Hampsey M. Molecular Genetics of RNAP. Microbiology and Molecular Biology Reviews p7.

89. Termination of Transcription
1. Intrinsic termination = termination sites
Terminator Sequence
Encodes the termination signal
In E. coli – base paired hair pin (rich in GC) followed by UUU…
causes the RNAP to pause
causes the RNA strand to detach from the DNA template

90. Termination of Transcription
2. Rho termination = Rho protein, ρ

91. prokaryotes: transcription and translation happen in cytoplasm
eukaryotes: transcription (nucleus); translation (ribosome in cytoplasm)

92. capping: guanylyl residue
In eukaryotes, mRNA is modified after transcription
Capping, methylation
Poly-(A) tail
splicing
capping: guanylyl residue
capping and methylation ensure stability of the mRNA template; resistance to exonuclease activity

93. Eukaryotic genes are split genes: coding regions (exons) and noncoding regions (introns)

94. Introns & Exons Intervening sequences Expressed sequences Introns

95. Splicing splicing occurs in the spliceosome!
Spliceosome: multicomponent complex of small nuclear ribonucleoproteins (snRNPs)
splicing occurs in the spliceosome!

96. EXERCISE
Enumerate all the enzymes and proteins involved in DNA replication and briefly state their importance/function. A short concise answer will suffice. (5 pts)
Give the partner or complementary strand of this piece of DNA:
5-ACTCATGATTAGCAG-3 (2 pts)
Provide the mRNA transcript of this DNA template strand:
5’-GGATCAGTAGCTAGCAGCTCGAGA-3‘ (4 pts)

97. Translation: Protein Synthesis
Central Dogma

98. Translation
Starring three types of RNA
mRNA
tRNA
rRNA

99. Properties of mRNA
In translation, mRNA is read in groups of bases called “codons”
One codon is made up of 3 nucleotides from 5’ to 3’ of mRNA
There are 64 possible codons
Each codon stands for a specific amino acid, corresponding to the genetic code
However, one amino acid has many possible codons. This property is termed degeneracy
3 of the 64 codons are terminator codons, which signal the end of translation

100. Genetic Code 3 nucleotides (codon) encode an amino acid
The code is nonoverlapping
The code has no punctuation

102. Synonyms Different codons, same amino acid
Most differ by the last base
XYC & XYU
XYG & XYA
Minimizes the deleterious effect of mutation

103. Practice Encoded sequences.
(a) Write the sequence of the mRNA molecule synthesized from a DNA template strand having the sequence
(b) What amino acid sequence is encoded by the following base sequence of an mRNA molecule? Assume that the reading frame starts at the 5 end.

104. Answers
(a) 5’ -UAACGGUACGAU-3’ .
(b) Met-Pro-Ser-Asp-Trp-Met.

105. tRNA as Adaptor Molecules
Amino acid attachment site
Template recognition site
Anticodon
Recognizes codon in mRNA

106. tRNA as Adaptor Molecules

112. Mechanics of Protein Synthesis
All protein synthesis involves three phases: initiation, elongation, termination
Initiation involves binding of mRNA and initiator aminoacyl-tRNA to small subunit(30S), followed by binding of large subunit (50S) of the ribosome
Elongation: synthesis of all peptide bonds - with tRNAs bound to acceptor (A) and peptidyl (P) sites.
Termination occurs when "stop codon" reached

113. Translation: Initiation
Translation occurs in the ribosome
Prokaryote START
fMet (formylmethionine) bound to
initiator tRNA
Recognizes AUG and sometimes
GUG (but they also code for Met
and Val respectively)
AUG (or GUG) only part of the initiation signal; preceded by a purine-rich sequence

114. Translation: Initiation
Eukaryote START
AUG nearest the 5’ end is usually the start signal

115. Elongation

118. Termination Stop signals (UAA, UGA, UAG):
recognized by release factors (RFs)
hydrolysis of ester bond between polypeptide and tRNA

119. Reference:
Garrett, R. and C. Grisham. Biochemistry. 3rd edition
Berg, JM, Tymoczko, JL and L. Stryer. Biochemistry. 5th edition