Mechanisms of Transcription Flashcards
RNA Pol I
transcribes the large rRNA gene
Types of RNA polymerase
- bacteria have one
- eukarya have up to 5
1. Pol I transcribes the large rRNA gene
2. Pol II transcribes mRNA genes
3. Pol III transcribes tRNA, snRNA 5s rRNA genes
4. Pol IV and V are found in plants and transcribe siRNA genes
RNA Pol II
transcribes mRNA genes
RNA Pol III
transcribes tRNA, snRNA 5s rRNA genes
Initiation
- RNA pol and other invitation factors recognize DNA segment
- RNA pol surrounds transcription bubble
- Transcription begins 5’-3’
- Only one strand acts as a template
Elongation
- After 10 bases into initiation, switch to elongation phase
- RNA pol steps along the DNA generating a mRNA copy of the template
Termination
Well defined in sim cell with specific sequences triggering termination
In others not so clear
RNA polymerase holoenzyme bacteria
Core enzyme
Sigma factors
Prokaryote preinitiation
There are consensus sequences in promoters that are recognized by specific σ factors
The degree of consensus corresponds with strength of promoter (how many transcripts per unit time).
Determining consensus in E. coli
σ70 recognizes promoters with 2 conserved sequences of 6nucleotides separated by 17-19 nucleotides
- 10 and -35 regions (6nucleotides)
- 10 (TATA BOX)
Variations on a theme consensus
σ70 recognized promoters
-35 (space 17-19bp) -10
Promoters for rRNA
UP-element -35 -10
Promoters for gal genes
Extended -10
dsDNA
Closed complex
Prokaryote initiation details
- RNA pol binds the DNA promoter in the closed complex (dsDNA)
- Isomerization: σ70 associates transition from closed to open complex (ssDNA) (pulls up an adenine and a Thyamine)
Prokaryote elongation details
- RNA pol must escape promoter
- Only synthesizes 9 or fewer nucleotides - abortive initiation
- Loss of σ is a necessary step
Elongation begins upon escape
Functions of RNA pol
- Catalyze RNA synthesis
- Unwind DNA
- Reanneal DNA
- Dissociate mRNA from DNA (only 8-9bp of mRNA remain base paired to DNA)
Types of proof reading by RNA pol
- Pyrophophorytic editing
2. Hydrolitic editing
Pyrophophorytic editing
- Uses the active site, backtrack 1 base
- Reincorporation of PPI
- Can remove correct or incorrect base
Hydrolitic editing
- Enzyme backtracks 1+ base
2. Cleaves RNA product, removing error
Termination in prokaryotes
Ends of genes have terminator sequence
- Rho-dependent
- Rho-independent
Rho-dependent termination prokaryotes
- Uses Rho factors (ATPase and helicase)
- Rut sequences in terminator
- ATP hydrolysis dependent
Rho-independent termination prokaryote
- Short inverted repeat and A-T region
- Transcription = stem loop in RNA
- Disrupts transcription - disrupts interaction between pol and DNA
Promoter eukaryote transcription
- Core promoter
- Proximal promoter
- Distal promoter
Core promoter
- Minimum portion of the promoter required to properly initiate transcription
- 40-60 Bo
- Has transcription short site (TSS)
- Binding site for RNAP
- General TF binding sites
Proximal promoter
- Sequences closer to the gene
- Tends to contain primary regulatory elements
- 250 bp upstream of the start site
- Specific transcription factor binding sites
Dealing with Histones
- FACT removes H2A-H2B dimer and holds onto it
- RNAP can then pass through
- FACT then places back dimer
Capping
mRNA is easily degraded so needs capping
The cap is a modified guanine added to 5’end between initiation and elongation
Capping steps
- RNA triphosphates cleaves gamma phosphate from 5’ end
- Guanyltransferase adds GMP to the terminal beta
- Methyltransferase adds methyl group to guanine
5’ cap serves to recruit ribosome to mRNA
What happens to CTD after capping?
- Dephosphorylation of ser-5 of RNAP II tail follows capping
- Allows capping machinery to dissociate
- New phosphorylation (ser-2) recruits splicing machinery
Polyadenylation steps
- Poly-A signal sequence is transcribed - leads to transfer of CstF and CPSF to mRNA and cleavage of mRNA.
- Poly-A polymerase adds 200 adenines to the 3’ end of mRNA - uses ATP as substrate but no template
- Poly-A binding proteins associate with mRNA - mature mRNA transported out of nucleus
Transcription termination - torpedo model
5’-3’ RNase (torpedo) recognizes uncapped end rapidly degrades RNA
Causes dissociation of RNAP II from DNA when encountered
Transcription termination - allosteric model
Dissociation of RNAP II from DNA occurs because RNAP II becomes less processive after poly-A site
5’-3’ RNase (torpedo) recognizes uncapped end and rapidly degrades RNA
Distal promoter
- Sequence farther from the gene
2. Weaker regulatory elements
GTF
General transcription factors
Preinitiation complex eukaryotes
- TATA box recognized by TFIID (complex with TBP and TAFs)
- Binding of TBP distorts TATA
- Distortion recruits TFIIA and TFIIB
- Recruit RNAP II and TFIIF
- RNAP II tail associates with GTFs
- TFIIE and TFIIH recruited last
- TFIIH mediates ATP hydrolysis leading to promoter melting
- Multiple rounds of abortive initiation follow
- Escape
Preinitiation complex escape
Phosphorylation of RNAP II tail (CTD) and ATP hydrolysis is needed for promoter escape
CTD
Carboxy-terminal domain (CTD) is the tail of RNAP II
Contains a series of heptapeptide repeats( Tyr-ser-pro-thr-ser-pro-ser)
Repeats contain phosphorylation sites targeted by TFIIH and others
CTD heptapeptide repeats
Tyr-ser-pro-thr-ser-pro-ser
In vivo preinitiation extra
Requires more complexes
- mediator complex
- activator : bring RNAP II and stabilize binding
- chromatin remodeling
- HAT
Elongation
- Shedding of initiation factors
- Recruitment of elongation factors
- Recruitment of RNA processing factors
- CTD tail transfers RNA processing factors to nascent mRNA
