Lecture 12

Question 1

Basic stages of transcription

Answer 1

• 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

Question 2

Prokaryotic RNA pols

Answer 2

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

Question 3

Explain prokaryotic promoters

Answer 3

• Consist of 2 DNA elements (for Sigma70)

-35 sequence - consensus sequence TTGACA

• 10 sequence (Pribnow box) - consensus sequence TATAAT

Question 4

Why study transcription?

Answer 4

Cancer
Autoimmunity
neurological disorders
Diabetes
Cardiovascular disease
Obesity

Question 5

What occurs with genes that are transcribed frequently?

Answer 5

They have promoter sequence more similar to consensus

Question 6

What do RNA polymerases have a greater affinity for?

Answer 6

Promoters with a sequence similar to the consensus

Question 7

What are the 3 types of eukaryotic RNA polymerase

Answer 7

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

Question 8

General transcription factors for RNA pol II

Answer 8

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

Question 9

Formation of pre-initiation comlpex

Answer 9

• 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

Question 10

What does TBP bind to?

Answer 10

Saddle shaped protein that binds minor groove

DNA bends by approx 80 degrees

Partial DNA unwinding

Question 11

What other genes on RNA pols is TBP important

Answer 11

tRNA and 5S RNA on RNA pol III

rRNA gene on RNA pol I

Question 12

Initiation next steps

Answer 12

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

Question 13

Abortive initIation

Answer 13

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

Question 14

Promoter clearance

Answer 14

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

Question 15

Biochemical reaction catalyzed by RNA pol

Answer 15

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

Question 16

mRNA processing

Answer 16

1. Transcriptional elongation coupled to mRNA processing in eukaryotes
2. Phosphorylation of 5th serine in heptad repeat of CTD region of RPB1 subunit occurs first
3. Binding of negative elongation factors which cause transcription pausing
4. RNA processing enzymes recruited by phosphorylation (guanosine cap added to 5’ end of mRNA)
5. Capping leads to phosphorylation of 2nd Serine in CTD heptad repear
6. RNA pol II resumes elongation

Question 17

How does elongation cause DNA supercoiling

Answer 17

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

Question 18

How do nucleosomes hinder eukaryotic RNA pol II?

Answer 18

Histone chaperones remove nucleosomes ahead of RNA polymerase and reassemble them behind the polymerase

Question 19

Examples of histone chaperones

Answer 19

ASF1, SPT6 and FACT

Question 20

Correcting mistakes

Answer 20

RNA polymerase stalls after encountering obstacles or problems

Reverses direction and most recently made RNA protrudes from complex

Transcript cleavage factors

Question 21

Transcription termination

Answer 21

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

Question 22

Termination by type II (allosteric model)

Answer 22

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

Question 23

Torpedo model

Answer 23

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