Lecture 9 - Transcription

Question 1

Key sites in prokaryotic RNA pol

Answer 1

• Beta subunit is the active site for RNA synthesis
• Sigma subunit fxns in promoter recognition and is not present during elongation (binds to -10 (TATA box) & -35 (CAT box)

Question 2

Steps in prokaryotic transcription

Answer 2

1. Polymerase binds nonspecifically to DNA & migrates along the molecule
2. Sigma subunit binds to the promoter
3. Polymerase unwinds DNA & initiates start of transcription
4. Sigma subunit is released
5. The growing mRNA chain is elongated

Question 3

Rho-independent termination of transcription

Answer 3

• because of palindrome in DNA, resultant single-strand RNA makes a hair-pin loop
• directly after this loop there are several A’s that get translated to U’s which causes the RNA pol to fall off DNA ending transcription

Question 4

Rho-dependent termination of transcription

Answer 4

• Rho protein binds to mRNA & moves up the mRNA & knocks RNA pol off DNA ending transcription

Question 5

Eukaryotic RNA pol types

Answer 5

• Pol 1: synthesizes rRNA
• Pol 2: synthesizes mRNA
• Pol 3: synthesizes tRNA and 5S RNA

Question 6

Eukaryotic conserved promoter consensus sequences

Answer 6

• -30 TATA box

- Inr (invariant region) built around +1 site

Question 7

Initiation of transcription in eukaryotes

Answer 7

• TBP (TATA binding protein) binds to -30 TATA box
• Remaining proteins in complex bind to form the closed complex
• DNA unwinds to form open complex
• RNA pol II is phosphorylated causing a conformational change that causes initiating proteins to fall off and elongation to begin

Question 8

Inhibitors of transcription

Answer 8

• Actinomycin D & Acridine: inhibit RNA elongation by intercalating into DNA template (Act D acts on DNA)
• Rifampicin: inhibits initiation of bacterial RNA synthesis by binding to the beta subunit of the polymerase (used in TB treatment)
• a-amantin: blocks eukaryotic pol II & pol III but not pol I or bacterial RNA polymerase (common toxin in poisonous mushrooms)

Question 9

5’ cap

Answer 9

• 7-methylguanosine
• co-transcriptionally added
• bond to mRNA is 5’-5’ bond
• protects message from degradation

Question 10

Poly-A tail

Answer 10

• mechanism by which eukaryotes stop transcription

Steps:

1. enzyme complex binds to cleavage signal sequence & displaces polymerase
2. RNA is cleaved 3’ of cleavage signal sequence (AAUAA)
3. Polyadenylate polymerase synthesizes poly-a tail

• some mRNA have longer poly-A tails
• the longer the poly-A tail the more stable the mRNA

Question 11

Non-self splicing introns

Answer 11

• found in most mRNA transcripts
• require snRNPs (small nuclear ribonucleoprotein)(RNA+protein)
• SLE (systemic lupus erythematosus: autoimmune disease where pt produces antibodies against snRNPs
• 5 snRNPs make a splicesome

Question 12

Splicing overview

Answer 12

• consensus sequences located @ splice sites
• splice donor site = 5’ splice site
• splice acceptor site = 3’ splice site
• branch site in middle of intron (always an A)
• forms lariat (see “lariat” think splicing mRNA)