chapter 4 dna, rna and the flow of genetic information
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Chapter 4 DNA, RNA and the Flow of Genetic Information. Backbone. Phosphodiester bond (-) charges protect “P” from being attacked by nucleophiles . Absence of 2’ –OH in DNA increase its resistance to hydrolysis Histones provide (+) charges for neutralization. Nucleic Acids. - PowerPoint PPT PresentationTRANSCRIPT
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