Chapter 4

DNA, RNA and

the Flow of Genetic Information

• DNA and RNA are unbranched linear polymers built up from similar units.

• Each monomer unit within the polymer consists of three components : a sugar,

a phosphate, and a base

Nucleic Acids

Backbone

• Phosphodiester bond

• (-) charges protect “P”

from being attacked by

nucleophiles.

• Absence of 2’ –OH in DNA

increase its resistance to

hydrolysis (akaline

conditions)

• Histones provide (+)

charges for neutralization.

Bases : Purines & Pyrimidines

A, G, C, T for DNA A, G, C, U for RNA

linkagewith sugar

throughN9

linkagewith sugar

throughN1

Nucleoside Phosphate; Nucleotide

Sugar-Base Linkages between

C1-OH of Sugars and N9 of Purines or

C1-OH of Sugars and N1 of Pyrimidines

Sugar-Phosphate Linkages between

C3-OH of Sugar and Phosphate or

C5-OH of Sugar and Phosphate

Nucleoside

Polarity of DNA Chain

• 5’ end OH is usually occupied by a phosphate group.• 3’ end OH is usually open as unmodified.• By convention, DNA base sequence is 5’ 3’ direction.• pACG ≠ pGCA

DNA Double Helix : Watson & Crick

• Right-handed coiled helix with two

polynucleotide chains

• Base : Inside

• Sugar & Phosphate : Outside

• Bases are perpendicular to the

helical axis.

• Adjacent bases separated by 3.4Å

• The same helical structure repeats

every 34Å.

• 10 bases per turn of helix

• Rotation of 36 degree per base

• Diameter of the helix : 20Å

• Hydrophobic interactions between

bases inside; hydrophilic polar

surfaceMaurice Wilkins, Rosalind Franklin – X-ray diffraction photograph of a hydrated DNA fiber

Watson-Crick Base Pairing in DNA by Hydrogen Bondings

Chargaff’s rule

1-5 kcalmol-1

• Under physiological condition, most DNA is in the B form.• A-form helix is less-hydrated DNA.

Z-DNA is a left-handed double helix in which backbone phosphates zigzag.

• Third type of DNA; From the structure of CGCGCG.• Left-handed, phosphates in the backbone zigzagged; called Z-DNA• Z-DNA-binding proteins required for viral pathogenesis.

Stacking of Base pairs for stability

• Hydrophobic effect just like protein • van der Waals forces - (0.5-1 kcalmol-1)

• π–π stacking

The Double helix facilitates the accurate transmission of Hereditary information

Semiconservative replication test by Dr. Meselson and Dr. stahl: upon DNA replication, one of the chains of each daughter DNA is newlySynthesized, whereas the other is passed unchanged from the parent DNA.

Complementary & Semi-Conservative DNA Replication

Genomic DNALabeling with

15NH4Cl Density Gradient

Centrifugationwith CsCl

Differ in density by about 1%

Melting & Annealing of DNA

Hypochromism by Base Pairing Denaturation of DNAby heat, acid or alkali

DNA sequence similarity between different organisms (i.e. relatedness) can be determined by the degree of hybridization.

Tm : the temperature at which half the helical structure is lost

Relaxed Circular DNA

Supercoiled Circular DNA

Linearity of DNA

Circular DNA vs. Linear DNA

Prokaryotic vs. Eukaryotic

Mitochondrial vs. Genomic

DNA Supercoiling

Helix vs. Superhelix

Topoisomerase

Structural Stability of Cellular DNA

Regulation of Gene Expression

DNA in vivo Packing Fold : 1000 times

DNA Topology

Complex Structures Formed by Single Strand Nucleic Acids

Stem-Loop (Hairpin)

Unusual

Base Pairing

between

Three Bases

(Long Range

Interaction)

Replication by DNA Polymerase

• Take instructions from templates (pre-existing DNA strands)

• DNA synthesis through complementary base pairing

• Step-by-step addition of deoxyribonucleotide to a DNA chain

(DNA)n + 5’-dNTP (DNA)n+1 + PPi

• Template

Primer strand with a free 3’-OH group

Nucleotides : dATP, dGTP, dCTP, TTP

Divalent metal ion : Mg2+

1. Complementary Base Pairing between template and incoming dNTP

(DNA Polymerase : Template-Directed Enzyme)

2. Nucleophilic attack by 3’-OH of the primer strand on the innermost phosphorous

atom of the incoming dNTP

3. Subsequent pyrophosphate (PPi) hydrolysis by pyrophosphatase provides further

driving force for the reaction.

4. DNA elongation direction : 5’ 3’

5. Exonuclease activity by DNA polymerase removes mismatched bases during

synthesis (3’5’) and after synthesis (5’3’) : proof-reading

(Error rate of DNA polymerase = 10-8 per base pair (1 억분의

DNA Polymerase Reaction

Some Viral Genomes Are Made of RNA

RNA Virus single-stranded RNA (viral genome) Protein

RNA-directed RNA polymerase for RNA replication

(e.g. Tobacco Mosaic Virus, influenza virus)

Retro-Virus single-stranded RNA (viral genome) RNA-directed DNA synthesis by

reverse transcriptase single-stranded DNA double-stranded DNA

integrate into the host genome replication together with the host

genome later, when it is necessary, express viral RNA and proteins

packaging virus particles and exit from the host (e.g. HIV-1)

Central Dogma : DNA RNA Protein

DNA ▪ storage of genetic information▪ serve only as information source during gene expression processes▪ minimize the chances of mutation

RNA ▪ photocopy of genetic information from DNA▪ dictate the repertoire of the proteins to be expressed (mRNA)▪ exist transiently as multiple copies (mRNA)▪ assist protein translation (rRNA, tRNA)▪ assist mRNA splicing and nuclear export (snRNA, hnRNA)

Crystal Structure of the large ribosomal subunit

•2009 Nobel prize in chemistry•Harry Noller at the University of California Santa Cruz•Venki Ramakrishnan at the University of Cambridge, •Thomas Steitz at Yale University

RNA Polymerase Reaction

• Complementary Base Pairing between DNA template and incoming NTP

(RNA Polymerase : DNA-Directed RNA Synthesizing Enzyme)

• Step-by-step addition of ribonucleotide to the RNA primer strand

(RNA)n + 5’-NTP (RNA)n+1 + PPi ; Primer is NOT required ;

Nucleotides : ATP, GTP, CTP, UTP ; Divalent metal ion : Mg2+, Mn2+

• Nucleophilic attack by 3’-OH on the phosphorous atom of the incoming NTP

• Subsequent pyrophosphate (PPi) hydrolysis

• RNA elongation direction : 5’ 3’ ; No proof-reading function

RNA Polymerases Take Instructions from DNA Templates

• Base Composition (Viral DNA vs. Viral RNA)• Hybridization Experiments between DNA template and transcribed RNA• Sequence Comparison between RNA and DNA templates

(template strand vs. coding strand; anti-sense strand vs. sense strand)

• Binding Sites for RNA Polymerase for Transcriptional Initiation

cf. TBP (TATA Binding Protein); TAFs (TBP Associated Factors); Basal Machinery

• Binding Sites for Various Transcription Factors for More Complex Regulation of

Transcription (Enhancer); quiet distant from the start site, on either 5’ or 3’ side

Promoter (Transcriptional Initiation)

Post-Transcriptional Modification of mRNA in Eukaryotes

5’ Capping & 3’ Poly-Adenylation

Terminator(Transcriptional Termination)

In Bacteria

1. Hairpin Forming Sequences & Poly-U Stretches

2. Transcription Termination Protein : rho

5’ capping structure;5’-5’ triphosphate linkage

Transfer RNAs Bring Amino Acids to the mRNA Template during Translation (Protein Synthesis by Ribosomes)

tRNA charged with amino acid : aminoacyl-tRNA : aa-tRNABy aminoacy-tRNA synthetase

Ester bond

mRNA protein: Adaptor molecule suggestion by Francis Crick

Codon in mRNA

Triplet Codons SpecifyEach Amino Acid

• Three nucleotides encode an amino acid.

• The code is non-overlapping.

• The code is sequentially translated without

punctuation.

• The genetic code is degenerated.

• 43 = 64 = 61 coding codons + 3 stop codons

• Codon degeneracy decreases the chances for

translational termination (64 = 20 + 44 ?).

• Codon degeneracy also reduces protein

sequence changes by genetic mutations.

• Codon degeneracy is most often found in

Wobble position (3rd base in a triplet codon)

• Recognition of stop codons by release factor

mRNA Contains Start and Stop Signals for Translation

• The first AUG (or GUG) is recognized by fMet-tRNA during translational initiation.

• Internal AUGs are recognized by Met-tRNA, and GUGs are by Val-tRNA.

• IF (initiation factor) vs. EF (elongation factor) for interactions with aa-tRNA

• Location of initiator AUG determines the reading frame for following triplet codons.

Formylmethionine Conjugated with

Initiator tRNA

Shine-Dalgarno sequence

The Genetic Code Is Nearly Universal

• The codon usage is almost invariant throughout the evolution.

• Translation of mRNAs from foreign species is usually successful.

• BUT, codon preference differs quite a bit between organisms.

• Mitochondrial gene expression utilizes slightly different codons.

(distinct set of tRNAs)

Mosaic Nature of Eukaryotic Genes : Introns & Exons

Exon: segments of nascent mRNA

retained in the mature mRNA

(usu. coding one domain)

Intron: segments of nascent mRNA absent

in the mature mRNADetection of

single-strandedDNA upon

DNA-mRNA hybridization

(electronmicroscopy)

Splicing

Typical Intron StructureBy spliceosome

Alternative Splicing

Allows Generation of

Protein Variants

from One Gene

Generation of Novel Genes by

Exon Shuffling during Evolution

Many Exons Encode Protein Domains

• Introns have been removed during evolution