Transcription

Question 1

Why can RNA transcription afford to be 100,000x less accurate than DNA replication?

Answer 1

RNA is temporary but DNA is permanent so consequence is less severe

RNAP can be less accurate than DNAP because RNA molecule is much shorter than DNA molecule (usually only 1000 bases)

Question 2

Describe RNAP in prokaryotes

Answer 2

RNA polymerase - PROKARYOTES

• multimeric
• large apoenzymes (cannot bind to DNA on its own) with 5 core sub units
• bacteria have a sigma unit (plus sigma makes it holoenzyme) which stabilises interaction with DNA
• 2 heterodimer beta subunits
• 2 homodimer alpha subunits - help polymerase bind to DNA
• Mg2+ in active site -> positive charge attracts negative charge of phosphate backbone

BACTERIAL RNAP

• B claws clamp down on DNA to melt it and produce transcription bubble with 2 single stranded regions
• rudder destabilises DNA/RNA hybrid and controls size of transcription bubble
• Alpha ubunits bind to DNA and stabilise it
• flap traps mRNA and protects it

ARCHAEA - RNAP
- have 1 RNAP that is similar to eukaryotic RNAP

Question 3

Describe RNAP in eukaryotes

Answer 3

3 Different Types specialised for diff types of RNA:

• rRNA - RNAP I
• mRNA - RNAP II
• tRNA - RNAP III
• have alpha subunits like prokaryotes but are heterodimers
• more complex with extra subunits
• NO sigma unit
• have long c-terminal domain tail
• lots of alcoholic -OH groups - provides H bonding opportunities for accessory proteins

Question 4

Describe transcription initiation in Bacteria

Answer 4

Transcription Initiation in Bacteria:

1. Initiation begins at conserved AT rich promoter sequences
2. bacterial RNAP binds directly to DNA via sigma subunit
   - diff sigma factors have diff specificities
   - Consensus sequence is optimal sequence for binding of sigma subunit of RNAP - increases frequency at which DNA is transcribed

• Pribnow Box - section RNAP binds to
• Stochastic, not regimented process; RNAP moves randomly, binds to diff sequences until consensus seq reached

Question 5

Describe transcription initiation in Eukaryotes

Answer 5

Transcription Initiation in Eukaryotes

transcription factors bind to promoter sequences

Basal transcription factor complex:
- TFIIH - composed of helicase and kinase - melts DNA and phosphorylates CTD of RNAP-II
- TFIIB - homologous to sigma factor - binds to B response element (BRE)
- TFIID - has TATA binding protein (TBP)
- etc
(ARCHAEA USE SIMILAR PROTEIN WITH TBP)

1. TBP binds to TBP binding region on sequence of DNA
2. TFIIB recognises and binds to BRE and recruits other factors to bind, eventually RNAP-II binds
3. TFIIH melts DNA and phosphorylates CTD of RNAP
4. RNAP-II begins transcription, leaves some transcription factors behind (unlike prokaryotes where sigma is part of RNAP)

Question 6

Describe transcription elongation in prokaryotes and eukaryotes

Answer 6

Transcription Elongation

Abortive Initiation: (to test the sites)

1. RNAP binds to promoter
2. sends out 8 nucleotide fragments
3. Falls off promoter OR goes through promoter clearance - depending on how well enzyme is interacting with DNA

IF RNAP sticking well to DNA → Promoter Clearance:

1. Flap of RNAP traps mRNA
2. Clamp traps DNA causing conformational change
3. IN Bacteria, sigma dissociates
4. Elongation begins
   Elongation is more involved in EUKARYOTES because nucleosomes must be remodelled

→ Histone acetyltransferase acetylates lysine and loosens histone binding to allow RNAP to better access DNA → moves more freely

= histone acetylation increases gene expression.

Question 7

How does transcription termination occur in eukaryotes?

Answer 7

Transcription Termination - Polyadenylation:
addition of lots of adenines to end of mRNA tail - post transcription modification

On mRNAs, the poly(A) tail protects the mRNA molecule from enzymatic degradation in the cytoplasm and aids in transcription termination, export of the mRNA from the nucleus, and translation

Question 8

How does transcription termination occur in prokaryotes?

Answer 8

Transcription Termination - ρ factor (Rho factor): hexameric helicase which ‘winds’ up mRNA and disrupts DNA/RNA hybrid in transcription bubble by physically getting in the way

• *Intrinsic Termination**
• does not require a special protein to signal for termination and is controlled by the specific sequences of RNA
• Self complementary sequence forms hairpin, disrupts RNAP’s flap causing it to stay open so mRNA is untrapped and free
• Hairpin formed from G-C bonds (stronger)