Genetic Code and Transcription
Central Dogma
• Central Dogma of Molecular Biology
• Genetic Code
– Triplet Code
– Degeneracy and Wobble
– Open Reading Frames
One Gene
• Transcription
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RNA Polymerase
Gene Structure
Three stages of transcription
Eukaryotic Transcription
Preinitiation Complex
mRNA Processing
One Polypeptide
Phenotype
• Translation
– Machinery
– Three stages of translation
– Prokaryotic vs Eukaryotic Translation
• Triplet Code
• Codon
• Special Codons
– Start Codon
– Stop Codons
• Degeneracy
• Wobble
5’
Open Reading Frames
ACUGUCGAUGUUCCACAACGGGAAGUGCUAAACUGUUGUCAUGAAA….
Met Phe His Asn Gly Lys Val (stop)
• Identify Start Codon
– Eukaryotes – First AUG
– Prokaryotes – after Shine
Dalgarno sequence
(AGGAGG)
• Start Codon Establish
Reading Frame
• Stop at stop codon
Transcription
RNA Polymerase
• Enzyme
• Synthesizes RNA by adding nucleotides to 3’
hydroxyl
• Substrate
NTP
• Requires
double stranded template
Mg++
No Primer
(Compare to primase)
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Gene Structure
Three Parts to Transcription
DNA
1. Initiation
RNA Polymerase binds DNA – determine by promoter
Short region double strand separated (transcription bubble)
First two nucleotide attached
2. Elongation
-1/+1
Promoter
Start Codon
Stop Codon
Additional Nucleotides added one at a time
RNA Polymerase moves down DNA
Transcription bubble moves with polymerase
Terminator
3. Termination
3’ UTR
5’ UTR
Triggered when polymerase reaches terminator
Start Codon
Stop Codon
Newly synthesized RNA released
RNA polymerase released from DNA
Transcription bubble closes
mRNA
Open Reading Frame
(ORF)
Prokaryotic Initiation
Prokaryotic Consensus Promoter
•
RNA Polymerase Structure
– Core Enzyme
– Sigma Subunit
• Holoenzyme
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Eukaryotic Initiation
• Three RNA Polymerases
– RNA PolI
– RNA PolII
– RNA PolIII
large rRNA
mRNA snRNA
tRNA 5SrRNA snRNA
RNA Polymerase binds Promoter
Release of Sigma subunit before
elongation
Alternative sigma subunits
Preinitiation complex
Assembly of complex
Role of Mediator
• Pol II Promoter
– Proximal and distal sequences
– TATA box most common pol II element
– Consensus ATATAA -35
2
5’ Cap structure
7 methyl guanosine triphosphate
mRNA Processing Eukaryotes
• Three types of processing
– 5’ CAP
– 3’ PolyA tail
– Spicing
Capping Enzyme in Nucleus
Protects RNA from 5’-3’ exonucleases
Promotes export from nucleus
Recruits Ribosome for Translation
PolyA tail
Splicing
Spicosome
Protein
snRNA
Remove introns that
interrupt orf
Alternative Spicing
Cleavage Complex
Poly A Polymerase
Bound by PolyA binding protein – protects from 3’-5’ exonuclease
Promotes Translation
Alternative Spicing
Translational Machinery
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tRNA
mRNA
Ribosome
GTP
Protein Factors
– Initiation Factors
– Elongation Factors
– Release Factors
3
tRNA
Charging tRNA’s
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Ribosomes
Three Stages of Translation
• Translational Initiation – 1 GTP
• Translational Elongation
• Delivery of charged tRNA – 1 GTP
• Transpeptidase Activity
• Translocation – 1 GTP
• Translational Termination – 1 GTP
20 Amino Acids
20 Synthetase
Cognate tRNA’s
2 ATP equivalents
tRNA cycle
tRNA binding sites
Translational
Initiation
• Small subunit and
initiator tRNA bind to
start codon. – tRNAi
is in P site
• Large subunit binds
clamping down on
mRNA
• GTP is hydrolyzed to
allow large subunit
binding
4
Elongation Cycle
1. Delivery of tRNA
2. Transpeptidase
3. Translocation
Polysomes
Translational
Termination
Prokaryotic vs Eukaryotic
• Prokaryotic transcription/translation coupled
• Eukaryotes transcription and translation are temporally and spatially
separated
Initiation
• Prokaryotes small subunit binds Shine
Dalgarno sequence (AGGAGG)
• Eukaryotes small subunit and tRNAi bind
cap and scan to first start codon.
# of ORFs
• Prokaryotic mRNA often polycistronic – more than one orf
• Eukaryotic mRNA usually monocistronic – one orf.
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