Transcription-general principles Flashcards
The mRNA sequence is the same as the…
Non-template strand/+ strand
What are the features of prokaryotic RNApol
One RNA pol that transcribes all RNAs
Core enzyme has 4 subunits – α2ββ’ – these catalyze RNA synthesis
ω subunit binds core and facilitates holoenzyme formation
σ factor binds core – σ factor directs polymerase to the promoter
What are the different prokaryotic sigma factors?
There are multiple σ factors in E. coli
- σ70 responsible for binding to most promoters in E. coli
- σ32 responsible for binding promoters of heat shock genes
- σ28 responsible for binding flagellin gene promoter
How does transcription begin?
RNApol binds to promoter
How is promoter strength determined?
Strong promoters are used frequently,
weak promoters are used less frequently. Strength is dictated by sequences at -35
and the Pribnow box
What is the -35 sequence?
sequence in the promoter 35 nts before start site
What is the Pribnow box?
– Pribnow box – occurs at -10 (i.e. 10 bp before the initiation site)
Where is the promoter located?
5’ end of the gene
What happens when RNApol holoenzyme binds to promoter?
Holoenzyme binds to promoter
- σ factor or β’ unwinds DNA at Pribnow sequence
- enzyme/promoter complex is now called “open”
Ribonucleotides can now base pair with the template strand
How does RNApol work?
RNA Pol catalyzes polymerization
- like DNA synthesis – mechanism is nucleophillic attack by 3’-OH
group on the α-phosphate on the following nucleotide
What is located at the 5’ end of a prokaryotic mRNA?
triphosphate
What must happen for transcription to proceed into the elongation phase?
RNA Pol must successfully synthesize an RNA ~10 nts long in order to
progress to the next phase
- if it fails – the little RNA is release and degraded – Pol tries again
- if it succeeds – conformation of Pol changes, σ factor is released
and proceeds to elongation
What is a transcription bubble?
Transcription bubble – segment of DNA that is temporarily single stranded during
transcription – this is 12-14 nts long
Note that RNA Pol covers ~30 nts – therefore transcription bubble protected
by RNA Pol
How is ssDNA protected during transcription?
Blocked by RNA pol
What is the channel structure of RNA polymerase?
RNA Pol has 3 channels – coding strand passes through one channel
- template strand passes through second channel – nucleotides bind
here and synthesis occurs
- RNA passes through a third channel to be released
The channels containing the coding and template strands end in proximity so the two strands can rapidly re-anneal
What enzyme relieves supercoiling tension during transcription?
topoisomerases
How does intrinsic termination work?
Intrinsic termination – termination sequence has an inverted repeat
followed by a string of U’s (in the RNA)
- this repeat can form a strong, stable hairpin
- hairpin stalls the RNA Pol
- U’s in the RNA base paired to A’s in the template strand are
weak
- RNA is released, transcription terminates
What is an inverted repeat?
5’ -> 3’ sequence on the template strand is the same as a 5- -> 3’ sequence on the non-template strand further downstream
How does rho-dependent termination work?
Rho factor – ATP-dependent helicase - binds a specific sequence in nascent RNA - translocates along RNA - unwinds DNA/RNA duplex - dislodges RNA Pol and transcription terminated
Believed to be a weak hairpin formed
downstream of rho binding site to stall
RNA Pol
How can histones be modified? What are the effects on transcription?
Histones can be posttranslationally modified, e.g. acetylation, methylation, phosphorylation
- modifies histone interactions to alter chromatin structure
- e.g. need histone acetylation for access to promoters
histone acetyl transferases (HATs) – promote gene expression
histone deacetylases (HDACs) – impair gene expression
How can protein complexes modulate histone interactions?
- SWI/SNF complexes promote histone release from DNA
- NURF proteins weaken histone/DNA interactions so they can slide
What RNAs do each of the 3 RNA polymerases in eukaryotes transcribe?
RNA Pol I – transcribes larger rRNAs
RNA Pol II – transcribes mRNA precursors, miRNAs and snRNAs
RNA Pol III – transcribes tRNA, 5S rRNA, U6 snRNA, snoRNAs
How do eukaryotic RNA pols differ from prokaryotic?
Eukaryotic requires additional transcription factors
What are the parts of eukaryotic promoters?
Three parts – core promoter – required for RNA Pol binding
- proximal promoter elements – transcription factors bind
- distal promoter elements – can be upstream or downstream
- function to enhance or repress transcription
- binding sites for enhancers or repressors
What are the two types of eukaryotic promoters?
focused promoter – has a major transcriptional
start site (TSS)
- dispersed promoter – multiple TSSs over 50-100 nts
What is a focused promoter? What is an example?
Set of core promoter elements
examples of CPEs – TATA box
- bound by TATA box binding protein (TBP)
- part of TFIID
- initiator (Inr)
- downstream of promoter element (DPE)
What are the characteristics of dispersed promoters?
Usually occur in CpG islands, and with housekeeping genes
What are examples of proximal promoter elements?
Proximal promoter elements – within about 250 bp of TSS
- examples – GC box – bound by transcription factor called SP1
- CAAT box -
Where can distal promoter elements be located? What are their functions?
Distal promoter elements – further away
- upstream, downstream or even in introns
- enhancers or repressors
What is the major subunit of RNA Pol II
RBP1
What are the features of RBP1?
RBP1 – has a C-terminal domain (CTD)
- has a repeated sequence YSPTSPS
- up to 52 repeats of the sequence
- site of posttranslational modification – phosphorylation
How does phosphorylation affect RBP1 activity?
unphosphorylated – binds promoter - Ser2 and Ser5 phosphorylation change to regulate switch to processivity histone remodelling 5’ cap formation splicing initiation
What 6 binding factors does RNA Pol 2 bind? Which is the major one?
TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH
- these facilitate assembly of pre-initiation complex
TFIID is major
What is the mediator complex?
Mediator - ~25 subunit protein complex
- binds to RNA Pol II and binds to enhancers and repressors bound to
distal promoter elements
What is the function of TFIIH factor?
TFIIH – helicase – ATP-dependent unwinding of DNA to make transcription bubble
What does RNA Pol II need to do to clear the promoter and proceed to elongation?
RNA Pol II needs to make 23 nts RNA for promoter clearance
- TFIIE, TFIIH release for clearance
- mediator release – go to elongation
What is the transcription termination sequence? What happens when it is recognized?
- AAUAAA
- this is recognized by several proteins bound to the CTD
- cleave transcript 10-30 nts downstream of this sequence
How is the poly-A tail generated?
Poly(A) polymerase – generates a poly(A) sequence 100-250 As long
- covalently linked to the 3’ end of the message
Are all mRNAs polyadenylated?
No
What are hnRNPs?
Pre-mRNAs associate with 20 different types of proteins to form hnRNP
- heteronuclear ribonucleoprotein particles
How is the 5’ end of pre-mRNA modified?
5’ end of pre-mRNA is modified – a 5’ cap is added
- 7 methyl guanosine is covalently linked
to the 5’ end via triphosphate linkage
- protection from nucleases
- ribosome recognition
What catalyzes splicing?
The spliceosome–a large RNA/protein complex
What sequences within the introns are recognized as splice sites?
GU-AG and AU-AC
What is the branch site?
Another important nucleotide in the intron is an A near the 3’ end of the intron.
This is called the branch site
What reactions occur at the branch site?
Two reactions occur: 1) 2’-OH of branch site A attacks the phosphate at the 5’ splice site – transesterification rxn - intron forms a lariat
2) Lariat is cleaved – 3’ OH of upstream
exon attacks P next to lariat
What is the result of the branch site reactions?
Exons are spliced together, intron is removed
What is an exon junction complex?
Complex that binds to mRNA 22 nts upstream of each exon junction
What is the function of EJCs?
Regulating nonsense mediated decay
If stop codons are found between EJCs, mRNA is degraded
