L12: Introduction to transcription

Question 1

Transcription: directionality; 1 key difference between replication

Answer 1

• RNA transcribed 5’ to 3’
• Template DNA strand read 3’ to 5’
• Doesn’t require a primer

Question 2

Transcription process in brief

Answer 2

1. Promoter sequence recognised and bound by RNA pol
2. Complex separates the two strands, first few nts synthesised
3. Once past the promoter, the complex undergoes conformational change
   -> interaction w/ DNA stabilised
4. Elongation continues until termination (specific sequence reached)

Question 3

RNA pols between organisms

Answer 3

• All have ‘crab claw’ structure, w/ AS where claws meet
• Analogous subunits found between bacteria, archaea and euk.
• Particular similarity between archaea and euk; bacteria slightly more simplistic

Question 4

Sigma factor

Answer 4

• Core enzyme: everything except sigma factor
• Holoenzyme: core enzyme + sigma subunit/factor (70 KDa)
• Locates the transcriptional start site by recognising promoter
  -> signals to start transcription
• C-terminal domain of alpha subunit also recognises upstream promoter element (UP)

Question 5

Conserved sites of promoter consensus

Answer 5

• -10, consensus sequence
• -35, consensus sequence, aka Pribnow box
  …(units upstream from start site)

Question 6

Different sigma factors in E.coli

Answer 6

• sigma D (normal - binds to housekeeping genes)
  Reponses to environmental change…
• sigma H (heat shock)
• sigma N (Nitrogen synthesis)
• sigma F (synthesis of flagella)

Question 7

Strong v weak promoters

Answer 7

• (Similarity of -10 and -35 to consensus sequence)
• Strong promoters: very similar; found in genes transcribed at very high rates; lots of mRNA required
• Weak: vice versa
  -> RNA pol has a much greater affinity for promoters of similar sequence to the consensus

Question 8

RNA pols in Eukaryotes

Answer 8

• Most have 3…
• RNA pol I:
  Found in nucleolus, rRNA synthesis, 28S, 5.8S rRNA found in large ribosomal subunit and 18S subunit
• RNA pol II:
  Found in nucleoplasm, synthesises mRNAs (protein coding), snRNAs (found in spliceosomes), miRNAs (regulate gene expression)
• RNA pol III:
  Found in nucleoplasm, synth. 5S rRNA, tRNAs (small adaptor molecules for placing aa in growing pp chain), snRNAs (small nuclear, found in spliceosomes)

Question 9

General transcription factors w/ functions (RNA pol II)

Answer 9

• TFIIA: stabilises binding of TBP and TFIIB
• TFIIB: promoter recognition, stabilise early transcribing complex
• TFIID: promoter recognition, DNA bending interacts w/ regulatory factors
• TFIIE: recruits TFIIH
• TFIIF: suppresses non-specific DNA binding, captures non-template strand upon melting
• TFIIH: unwinds promoter DNA, phosphorylates CTD

Question 10

TBP

Answer 10

• Tata box binding protein
• Binds tata box and recruits TFIID to the BRE upstream
  -> Remaining components then bind to form pre-initiation complex
• The TBP bends structure of DNA; AT rich regions particularly susceptible to deformation (H bond density low)

Question 11

Formation of the transcription bubble + abortive initiation definition

Answer 11

• All RNA pols open up about 14bp of duplex DNA
• In pol II, bubble opened up by helicase subunits of TFIIH (requires ATP - unique)
• Abortive initiation: When RNA pols fail to make a full length RNA on first attempt; leads to release of short RNAs of 2-9 nts

Question 12

Abortive initiation (process)

Answer 12

• Bacterial sigma factor and euk TFIIB involved in abortive initiation - each have loop that extends into AS region
• Loop is in a position to block elongating transcript so must be moved in order for transcription to occur
  -> displacement of protein loop allows promoter clearance
  -> more tightly bound to DNA, loosened grip on initiation factors

Question 13

Special features of RNA pol II

Answer 13

• Makes mRNA, but not usually in its final active form; ‘pre-mRNA’
• Transcriptional elongation is coupled to mRNA processing in eukaryotes
• Pol II is phosph. as it converts to the elongating complex
• Phosph. of the 5^th serine in the heptad repeat on the CTD region of Rpb1 subunit occurs first
  -> RNA-processing enzymes recruited, guanosine cap added to 5’ end of mRNA
  -> pausing of elongation
• Leads to phosph. of second serine in CTD heptad repeat, causes polymerase to resume elongation

Question 14

Stalling

Answer 14

• As transcription bubble moves along DNA, positive supercoiling increases ahead and negative supercoiling increases behind
  -> energetic barrier, stalling of RNA pols
• Tension must be relieved by topoisomerases
  e.g. DNA gyrase and DNA topoisomerase I in E.coli

Question 15

Dealing with chromatin; eukaryotic RNA pols

Answer 15

• Eukaryotes use histone chaperones to remove nucleosomes ahead of RNA pol, reassembling them behind the pol
  e.g. FACT (Facilitates Chromatin Transcription), Asf1, Spt6

Question 16

Stalling due to miscoding

Answer 16

• When the RNA pol stalls, this is detected
• Reverses direction, most recently made RNA protrudes from complex
• Transcript cleavage factors chop this off through stimulating RNA pol’s endonuclease activity
  -> correction

Question 17

2 examples of terminators in E.coli

Answer 17

1. Type I: aka Rho-independent/Intrinsic terminators. No additional factors required
   -> forms hairpin structure in RNA formed, followed by string of uridines
   -> RNA dissociates as a result, tr. stops
2. Type II: aka Rho-dependency. Rho factor required for termination (uses ATP)
   -> hexameric Rho uses ATP as a co-factor causing dissociation
   - Approx 50:50 in E.coli

Question 18

Allosteric model for termination by RNA pol II

Answer 18

• Most euk mRNAs have a poly-A tail added through polyadenylation
• RNA pol II continues to transcribe after the polyadenylation signal, but mRNA is cleaved here and the 3’ end of the mRNA is processed

Question 19

Torpedo model for termination by RNA pol II

Answer 19

• After cleavage, the RNA downstream of the poly(A) site is digested by a 5’ to 3’ ribonuclease (Rat1)
• Continues to degrade nascent RNA until it runs into RNA pol
  -> Disrupts polymerization and causes the enzyme to dissociate from the DNA