Transcription

C483 Spring 2013

1. RNA Polymerase

A. Polymerizes RNA in a 3’5’ direction.B. Has 3’5’ exonuclease activityC. Has 5’3’ exonuclease activityD. Catalyzes a reaction driven by pyrophosphate hydrolysis

2. Which of the following statements is true?

A. The template strand of DNA is copied by RNA polymerase 3’5’.B. The “sense” strand is DNA; the “antisense” strand is RNA.C. The coding strand of DNA is identical to the transcribed RNA.D. The template strand of DNA is identical to the transcribed RNA.E. A gene is read in the 3’5’ direction.

3. The rate limiting step in transcription is

A. Promoter bindingB. InitiationC. ElongationD. Termination

4. Weak promoters containA. no consensus sequences.B. sequences that match consensus sequences poorly.C. no TATA box.D. a TATA box but no -35 region.

5. Which of the following is not a difference between bacterial and eukaryotic transcription?

A. Bacterial transcription has less types of RNA polymerase.B. Eukaryotic transcription makes use of more general transcription factors.C. Bacterial mRNA undergo extensive post-translational modifications.D. Chromatin plays a large role in regulation of transcription in eukaryotic

cells.

Transcription• Gene—a DNA sequence that is transcribed • Housekeeping vs. regulated genes• Many types of RNA

Information Flow

• Central Dogma: information in protein cannot be incorporated into information in DNA

• RNADNA in retroviruses• We will focus on

messenger RNA (mRNA)

RNA Synthesis

• Definition of transcription: DNA-directed RNA synthesis

• RNA Polymerase• Three stages– Initiation– Elongation– Termination

Bacterial RNA Polymerase

• Multisubunit• Smaller than

replisome• Core enzyme• Plus s-subunit for

initiation

Chain Elongation Reaction

• Very similar to DNA polymerization

• Interacts with the DNA template over a short distance, then DNA double helix reforms

• No 3’5’ exonuclease

Genes• 5’ 3’ (downstream and upstream)• Coding strand vs. template strand• Sense strand vs. antisense strand

Initiation• Promoter—region of DNA that serves as

site for transcription initiation• Consensus region– TATA box– -35 region

• Strong promoter matches consensus region better—efficient transcription of rRNA

• Weak promoters—infrequent transcription

Initiation• Promoter binding is relatively fast and selective• role of s subunit– Decreases selectivity for nonspecific sequences

• Initiation is slow step– DNA unwinding– Synthesis of primer

• Elongation is fast

Nonspecific binding

One-dimensional promoter search

Holoenzyme and promoter form closed complex

Open complex allows initiation

Sigma subunit released; promoter is cleared and elongation starts

Termination

• Termination sequences– Pause site– Rho dependent

Differences in Eukaryotic Transcription

• Multiple RNA polymerases based on compartment and job

Differences in Eukaryotic Transcription

• General Transcription Factors—complex promoter binding

Differences in Eukaryotic Transcription

• Role of Chromatin– 35% of DNA is transcribed– Most of it is quiescent in any given cell– Buried in chromatin– Nontranscribed genes are relatively inaccessible– “Remodelling” enzymes make genes accessible

(for example, HATS)

Differences in Eukaryotic Transcription

• mRNA processing• Makes primary transcript into “mature”

transcript• PolyA tail makes RNA less susceptible to

exonucleases• “Splicing” of exons together, with removal of

introns– Mature RNA much shorter than primary transcript

Answers

1. D2. A3. B4. B5. C