transcription 1

Question 1

What are the three steps of transcription?

Answer 1

Initiation, elongation, termination.

Question 2

How can the chromatin be opened up for transcription?

Answer 2

A) Nucleosome remodeling enzymes: move the histone from one part of the genome to another.

B) Histone chaperones: disassemble and reassemble the histones.

C) Enzymatically-driven histone modification: Acetylation, methylation, ubiquitination, sumoylation, phosphorylation, and adenosine diphosphate (ADP) ribosylation

Question 3

What are the different RNA-polymerase classes?

Answer 3

• RNA polymerase I (Pol I) transcribes large ribosomal RNA (rRNA) genes
• RNA polymerase II (Pol II) transcribes messenger RNA (mRNA) genes
• RNA polymerase III (Pol III) transcribes a variety of RNAs including transfer RNA (tRNA) and 5S ribosomal RNA

Question 4

How is the bacterial RNA-polymerase constructed?

Answer 4

• 2 alpha subunits (1 NTD and one CTD domain)
• 2 beta subunits
• 1 omega subnits

Question 5

What is a holoenzyme?

Answer 5

The RNA polymerase form that is competent to initiate (de novo) transcription.

Question 6

What are the sigma domains?

Answer 6

Bind sigma factors, determines promoter specificity.

Question 7

What is the C-terminal domain (CTD)?

Answer 7

The domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins and UP-element.

Question 8

How are promoters recognized in bacteria?

Answer 8

By binding sigma factors to consensus sequences between a -35 and -10 element upstream of the TSS.

The closer the sequence resembles the preference for the sigma factor, the better the binding.

Question 9

(bacteria) How are sigma factors regulated? Why are they?

Answer 9

Pro-sigma factors = need to be cleaved before they can act with the core enzyme and function properly.

Anti-sigma factors = prevents binding of sigma factors to the DNA.

Can be regulated to prevent certain genes from being transcribed too early, ex. flagella genes.

Question 10

(eukaryotes) What are general transcription factors?

Answer 10

Extra proteins that assemble in large complexes with the RNA polymerase to create the pre-initiation complex.
For RNA polymerase II –> TFII

Question 11

(eukaryotes) How is the transcription initiation complex assembled?

Answer 11

• TFIID binds the TATA-box via TBP, causing unwinding
• TAFs recognize other promotor elements (INR, DPE)
• TFIIB is recruited, recognized BRE promoter element
• TFIIA binds, stabilizing binding between TBP and DNA
• TFIIE binds
• TFIIH binds and unwinds DNA with ATP-power

Question 12

What is Mediator?

Answer 12

A complex required to activate many genes transcribed by RNA pol. II.

Question 13

What is abortive initiation?

Answer 13

Is the RNA polymerase fails to make a full length DNA on the first attempt, it releases small 2-9 nucleotide sections.
This happens if the loop in sigma or TFIIB does not perform a conformational change to move out of the way, allowing the polymerase to clear the promoter.

Question 14

What is transcriptional pausing, and how can this be lifted?

Answer 14

The polymerase stops in the middle of RNA synthesis, usually because of physical obstruction.

Can be relieved or enhanced by elongation factors.
The polymerase can then backtrack, using transcript cleavage factors + endonuclease to remove the faulty sequence and start once again.

Question 15

(eukaryotes) How is pre-mRNA processed?

Answer 15

The CTD region of the Rpb1 subunit is phosphorylated.

• -> Recruits RNA-processing enzymes
• -> Caps 5’ end.

Question 16

(bacteria) How is transcription terminated?

Answer 16

Terminator sequences in the DNA.

Intrinsic terminators:

• have an inverted repeat sequence –> forms stem loop
• have a string of 8-10 A-residues –> causes instability

Enzymatic terminators:
- Rho-protein –> ring protein drives polymerase away from RNA by ATP-hydrolysis

Question 17

(eukaryotes) How is transcription terminated? (2 models)

Answer 17

A poly-A-tail is transcribed.

Allosteric model:

• RNA pol II transcribes through poly-A tail
• RNA processing proteins bind CTD
• Binding/cleaving the proteins gives conformation changes and dissociation of RNA pol II from DNA.

Torpedo model:

• The RNA downstream of the poly-A is digested by endonuclease
• When the nuclease hits the polymerase, it pushes it away
• The RNA pol II dissociates from the DNA