Unit 5: Transcription

Question 1

What are the differences between DNA replication and RNA synthesis?

Answer 1

• RNA transcription, not all DNA is transcribed
• Occurs during the whole cell cycle, not only during cellular division
• No primer needed to initiate
• Only 1 strand is synthesized

Question 2

What does transcription require?

Answer 2

A DNA template, a DNA-dependent RNA polymerase, ribo nucleotides, Mg2+

Question 3

What makes up the bacterial RNA polymerase core?

Answer 3

5 subunits + sigma factor

Question 4

What are the steps of transcrption?

Answer 4

1. Binding of RNA polymerase core to the DNA promoter
2. Formation of transcription bubble
3. Initiation
4. Elongation
5. Termination and recycling

Question 5

What is the role of the sigma factor?

Answer 5

RNAP core cannot effectively bind promoter without a sigma factor. There are different sigma factors specific to different promoters

Question 6

What are the steps for DNAse foot-printing assay?

Answer 6

1. Copy of the DNA (PCR)
2. Label 1 end with radioactivity
3. Add protein
4. Add DNAse (in correct amount so that it cuts each DNA only once, in random place)
5. Digest proteins and run in gel

Question 7

How does initiation go from a closed complex to an open complex?

Answer 7

1. RNAP binds to promoter -> closed complex
2. Spontaneous Isomerization –> open complex
   - pincers close around DNA downstream from TSS
   - N term of sigma 70 moves away from active-site cleft allowing DNA to take its place

Question 8

What is the difference between sigma 70 and sigma 54?

Answer 8

Sigma 70 does not require ATP hydrolysis, while sigma 54 does

Question 9

How does the synthesis of transcription occur?

Answer 9

RNAP needs to hold in place first 2 nucleotides to catalyze phosphodiester bond, during first bonds there is a high probability that RNAP will release transcript from template. If holoenzyme doesn’t release transcription at this stage, it repeats intiation

Question 10

When is the RNAP elongation phase started?

Answer 10

For transcription >10nt: structural transition to unblock RNA exit channel

Question 11

What do Rho-independant termination sequences have?

Answer 11

1. self-complementary 15-20 nt region that hair pins after exit from RNAP; causes RNAP to stall
2. AAA sequence downstream of hairpin that melts from RNA UUU during hairpin-induced stall

Question 12

What does Rho-dependent termination do?

Answer 12

Once helicase binds to RNAk it will quickly move and collapse onto polymerase causing the RNA to fall

Question 13

Why do eukaryotic polymerases require TF?

Answer 13

• more transcriptional control (different cell types)

- access to chromatin with nucleosomes

Question 14

What does RNA Pol 1 do? Where is it found?

Answer 14

Synthesis of large ribosomal RNA precursors. Located in nucleolus. Accounts for most of the RNA synthesis

Question 15

What does RNA Pol II do? Where is it found?

Answer 15

Synthesis of nearly all protein coding genes. Located in nucleoplasm. Accounts for greatest variety of the RNAs

Question 16

What does RNA Pol III do? Where is it found?

Answer 16

Synthesis of variety of small RNAs. Located in nucleoplasm

Question 17

What are the common type of promoters recognized by the polymerases?

Answer 17

TATA-binding protein

Question 18

What are the steps of transcription of Pol II?

Answer 18

1. General TFs bind promoter and recruit Pol II
2. DNA unwinding
3. At the end of initiation step: phosphorylation of C-terminal domain releases TF
4. Transcription terminates and CTD is dephosphorlyated
5. Release of RNA & RNAP