Lecture 12 Flashcards
Basic stages of transcription
- RNA transcribed 5’ to 3’
- First step - recognition + binding of promoter sequence by RNA pol
- DNA strands separated by enzyme complex
- Transcription initiates - first nucleotides of transcript synthesised
- Complex stable with DNA through conformational change after passing promoter
- RNA pol elongates RNA transcript until particular DNA sequence reached cause RNA pol to be released from template
Prokaryotic RNA pols
Core enzyme ~400kDa
5 polypeptides - 2 copies of alpha subunit, one of beta’, beta, and w
Sigma factor - 70kDa
Recognises promoter - Allow RNA pol binding
Holoenzyme
Core enzyme + sigma factor
Explain prokaryotic promoters
- Consist of 2 DNA elements (for Sigma70)
-35 sequence - consensus sequence TTGACA
- 10 sequence (Pribnow box) - consensus sequence TATAAT
Why study transcription?
Cancer
Autoimmunity
neurological disorders
Diabetes
Cardiovascular disease
Obesity
What occurs with genes that are transcribed frequently?
They have promoter sequence more similar to consensus
What do RNA polymerases have a greater affinity for?
Promoters with a sequence similar to the consensus
What are the 3 types of eukaryotic RNA polymerase
RNA polymerase I - Nucleolus
Contains rRNA - 28S, 5.8S large ribosomal unit and 18S small ribosomal unit
RNA polymerase II - Nucleoplasm
mRNA - protein coding
snRNAs - Small nuclear RNA found in spliceosomes
miRNAs - micro RNA - regulates gene expression
RNA polymerase III - Nucleoplasm
5S rRNA - component of large ribosomal unit
tRNAs - Small RNA adapter molecules - place amino acid in growing polypeptide chain
snRNAs - small nuclear RNAs found in spliceosomes
General transcription factors for RNA pol II
TFIIA - 3 - Stabilizes binding of TBP and TFIIB
TFIIB - 1 - Promoter recognition
TFIID - 15 - DNA bending interacts with regulatory factors
TFIIE - 2 - Recruits TFIIH
TFIIF - 2 - Suppresses non-specific DNA binding
TFIIH - 9 - Unwinds promoter DNA
Formation of pre-initiation comlpex
- Core promoter elements assemble transcription initiation complex
- TBP binds TATA box - Promotes binding of TFIIB to BRE sequence
- Remaining components bind to form pre-initiation complex
What does TBP bind to?
Saddle shaped protein that binds minor groove
DNA bends by approx 80 degrees
Partial DNA unwinding
What other genes on RNA pols is TBP important
tRNA and 5S RNA on RNA pol III
rRNA gene on RNA pol I
Initiation next steps
RNA pols open 14bp of duplex DNA (transcription bubble)
In pol II, transcription bubble opened by helicase subunits of TFIIH which requires ATP
RNA pols fail to make full length RNA on first attempt
Abortive initiation leads to release of short RNAs of 2-9 nucleotides
Abortive initIation
Bacterial sigma factor and eukaryotic TFIIB involved in abortive initiation - have loop that extends into active site region
Loop block elongating transcript, so is moved for transcription to continue
Promoter clearance
Displacement of protein loop helps polymerase break away from promoter - promoter clearance
RNA pol undergoes conformational change associating it stably with DNA, and loosens grip on initiation factors
Biochemical reaction catalyzed by RNA pol
alpha phosphate undergoes nucleophilic attack by 3’ OH group of last nucleotide in growing RNA chain
Release of resulting pyrophosphate is hydrolysed to yield monophosphate, generates energy for reaction
mRNA processing
- Transcriptional elongation coupled to mRNA processing in eukaryotes
- Phosphorylation of 5th serine in heptad repeat of CTD region of RPB1 subunit occurs first
- Binding of negative elongation factors which cause transcription pausing
- RNA processing enzymes recruited by phosphorylation (guanosine cap added to 5’ end of mRNA)
- Capping leads to phosphorylation of 2nd Serine in CTD heptad repear
- RNA pol II resumes elongation
How does elongation cause DNA supercoiling
Positive supercoiling increases ahead of polymerase, negative increases behind
Changes in supercoiling stall RNA pols (relieved by topoisomerases)
DNA gyrase removes positive supercoils, DNA topoisomerase I removes negative supercoils
How do nucleosomes hinder eukaryotic RNA pol II?
Histone chaperones remove nucleosomes ahead of RNA polymerase and reassemble them behind the polymerase
Examples of histone chaperones
ASF1, SPT6 and FACT
Correcting mistakes
RNA polymerase stalls after encountering obstacles or problems
Reverses direction and most recently made RNA protrudes from complex
Transcript cleavage factors
Transcription termination
Type I terminators - Rho-independent or intrinsic terminators - no additional factors required for termination
Type II terminators - Rho-dependent terminators - Rho factor required for termination (uses ATP)
Approx half of E. coli transcripts terminated at each type of terminator
Termination by type II (allosteric model)
RNA pol II transcribes through polyadenylation and 3’ end processing signals
RNA processing complexes associate with both the processing signals phospotylated CTD
mRNA cleaved
Transcription terminated
RNA pol II released
Torpedo model
mRNA cleaved at polyA site
Nascent downstream of polyA cleavage site is digested by a 5’ to 3’ ribonuclease (Rat1)
Polymerization is disrupted and RNA pol II dissociates from DNA template
