RNA biology lecture 1

Question 1

Transcription initiation

Answer 1

• RNA pol = recruited to Pol promoter w/ TF
• RNA pol II TF assoc at TATA promoter via TBP, then TFIIA/B bind, Pol recruited w/ TFIIF/H
• RNA pol transcribes gens in 5’-3’ direction to form pre-mRNA

Question 2

Prokaryotic vs eukaryotic transcription

Answer 2

• Eukaryotic transcrip/translat occur in different compartments
• Unlike bacteria x have co-transcription
• Means RNA potentially exposed to exo-ribonucl.
• Recruitment of ribosomes in prokaryotes = controlled by 5’ SD
• Eukaryotes x have SD
• Eukaryotic genes = interrupted coding sequences

Question 3

Pre-mRNA processing

Answer 3

• co-transcriptional
• Processing largely complete b4 termination, tight interconnection
• Different processing linked to different stages of transcription (capping early, splicing early + late, 3’ end late)

Question 4

Evidence - pre-mRNA processing

Answer 4

• All 3 occur in vitro w/o transcription
• Some components of processing interact w/ general TF, recruited to PIC
• Transcription activators indirectly influence efficiency of processing reactions
• Some factors disengage w/ TF + attach to polymerase
• So some factors recruited early
• Promoter swap experiment (GOI engineered so promoter specificity for recruitment of Pol replaced by sequence that recruits RNA Pol I) → pre-mRNA that x be processed
• CTD of polII links mRNA processing to transcription, deletion has ↑ effect on pre-mRNA processing

Question 5

CTD of RNA polII

Answer 5

• Composed of random heptad repeated motifs: YSPT SPS
• Ser2,5,7 = substrate for phosph by RNAPII
• Typically unphosph, Ser 5 phosph by Cdk7 of TFIIH
• Later in elongation, serine 2 phosph
• Phosph of CTD = dynamic
• Kinases (2 main kinases = cdk7 (phosph ser 5) + cdk9 (phosph ser 2)
• CTD phosphatases (reverse phosph in position dependent manner, Fcp1 targets Ser5/2, SCP dephosph Ser5)

Question 6

Phosph status of CTD changes along gene

Answer 6

• Follow Pol w/ ChIP, use Ab that recognise Ser2/5
• Phosph at Ser5 at start, peaks early in transcription cycle then ↓
• Opposite of Ser2 (↓ Ser5 corresponds ↑ Ser2 which peaks at end of transcription then ↓)
• At TSS, low phosph, at end FCP-1 dephosph
• CTD provides code
• Dynamics ↑ complex, Ser5 phosph linked to splicing, kinases/phosphatases x known