Gene Expression and RNA Processing Flashcards
What are the subunits of E. coli RNA Polymerase?
Alpha Beta Beta' W o70
Alpha subunit of RNA Polymerase
Required for assembly of core enzyme, interacts with regulatory factors (2 subunits)
Beta subunit of RNA Polymerase
Takes part in all stages of catalysis
Beta’ subunit of RNA Polymerase
Binds to DNA, takes part in catalysis
W subunit of RNA Polymerase
Required to restore denatured polymerase to its native form
o70 subunit of RNA Polymerase
Takes part in promoter recognition
Core enzyme + o70 = holoenzyme
Initiation of Transcription
- Sigma (o) subunit decreases the affinity of RNA polymerase for DNA
- Sigma subunit recognizes promoter sequence (-35, -10)
- After several nucleotides of RNA are synthesized, o falls off
- No primer required
Elongation Reaction for RNA Transcription
3’OH acts as nucleophile and attacks P
- PPi released, H2O added = 2Pi
Termination of Transcription
- Stemloop causes RNA polymerase to pause
- rU-dA base pairs are weak, so that RNA dissociates from the DNA template and enzyme
- Protein-dependent termination (Rho protein)
Transcription in Prok and Euk have what in common?
In common:
- NTP + RNAn + H2O -> RNAn+1 + 2Pi
- 3’OH group of the growing RNA chain attacks the innermost phosphate of next NTP
- RNA is formed in the 5’-3’ direction
Transcription in Prok vs. Euk differences
Prok differences:
- Transcription and translation occur at same time
- Simple control elements
- Termination signal is GC rich hairpin-poly(U) structure
- mRNA is not transported across a membrane
Euk differences:
- Transcription and translation are separated in time and space
- More complex control elements
- Termination signal is poly(A) tail
- mRNA is highly processed and transported across the nuclear envelope
Eukaryotic cells use three RNA polymerases to synthesize RNA:
mRNA, rRNA, tRNA
Cis-acting elements
DNA sequences that regulate expression of a gene located on the same molecule of DNA
Where is the TATA box located?
-25
Inr
Initiator element +1
Enhancer
Can be >1kilobases from start site
DPE
Downstream core promotor element +30
CAAT box and GC box
-40 to -150
Pre-Initiation Complex (PIC)
- Transcription factor bind cis-acting elements to recruit RNA pol II
- TATA box is recognized by TFIID by the TATA Binding Protein (TBP)
- TFIID is a dynamic protein complex
Important stuff to know for formation of the pre-initiation complex
1) TFFID binds DNA and the TBP domain, until eventually binds TATA box
4) TFIIB recognizes TBP/DNA complex and RECRUITS POL II!
5) TFIIH (helicase) unwinds DNA and PHOSPHORYLATES THE C-TERMINAL DOMAIN OF POL II (TRIGGER TRANSITION TO ELONGATION!)
Additional Transcription Factors Regulate Transcription
- PIC initiates transcription at low levels
- Additional TFs that bind to other sites stim high lvls of transcription (expressed in a tissue-specific manner)
- 1 TF has little influence, many are needed to form a complex that stims or suppresses transcription (Combinatorial control!)
mRNA Processing
- mRNA transcript is modified by 5’ cap and poly (A) tail
- Introns are removed and exons are joined during RNA splicing
During formation of the 5’ Cap
1) Removal of a phosphoryl group from 5’ triphosphate
2) Diphosphate 5’ end attacks alpha-phosphorous of GTP
3) N-7 of terminal guanine is methylated. Adjacent riboses may also be methylated by guanylmethyltransferase
Why do mRNAs have a 5’ cap?
Protection from degradation
