structure and function of the bacterial RNAP

Question 1

what is meant by operons?

Answer 1

genes encoding enzymes that are part of the same metabolic pathway are clustered close together and regulated by the same promoter

Question 2

how far apart are stacked base pairs?

Answer 2

3.4A

Question 3

how long is a hydrogen bond?

Answer 3

2.8A

Question 4

why does almost 100% of prokaryotic DNA code for proteins?

Answer 4

prokaryotic DNA is not processed and contains no introns

Question 5

why is E.coli useful?

Answer 5

• plasmids and cloning
• model systems
• biofuels
• antibiotic production and antibiotic resistance
• no ethical issues

Question 6

what are the steps in discovering an enzyme?

Answer 6

• make cell lysate
• invent a transcription assay; add 4NTPs, P32 ribonucleotide, DNA template, heat to 37 degrees for 30 minutes
• separate free NTP from radiolabelled NTP using ethanol
• purify using chromatography

Question 7

which subunits does bacterial RNA polymerase consist of?

Answer 7

A-subunit, B-subunit, B’-subunit, w-subunit

Question 8

what do the B and B’ subunits do?

Answer 8

contribute to RNA catalysis in active site

Question 9

what does the antibiotic microcin do?

Answer 9

blocks NTP entrance channel

Question 10

what is the role of the core enzyme?

Answer 10

synthesises RNA on a DNA template, but cannot recognise promoters

Question 11

what is the role of the sigma factor?

Answer 11

ensure initiation of transcription from specific sites, but also reduces binding to non-specific sequences

Question 12

how is the catalytic site formed?

Answer 12

formed by B and B’. has one Mg++ tightly bound to the enzyme in the active site, held in place by an ionic bond in coordination with oxygens and one of 3 aspartate residues

Question 13

how does one promoter control synthesis of more than one protein?

Answer 13

there are several open reading frames

Question 14

what are the steps for a transcription assay to discover an RNA polymerase?

Answer 14

1- make a cell lysate
2- add NTPs and a P32 labelled UTP, add a DNA template and heat to 37 degrees for half an hour
3- separate free NTP from radiolabelled NTP using ethanol
nucleic acids precipitate, but free nucleotides dont

Question 15

what does the A subunit of bacterial RNA polymerase do?

Answer 15

responsible for enzyme assembly, promoter recognition and binding to activators

Question 16

what is the role of the a and sigma subunits?

Answer 16

form the major surfaces on RNA polymerase for interactions of the enzyme with factors that regulate initiation of transcription

Question 17

where is the channel for DNA?

Answer 17

at the interface of the B and B’ subunits, these stabilise the separated single strands in the transcription bubble

Question 18

why does the core enzyme have general affinity for DNA?

Answer 18

electrostatic interactions between the protein and the DNA

Question 19

what can and can’t the core enzyme do?

Answer 19

can: synthesise RNA on a DNA template
cant: recognise promoters

Question 20

what does sigma factor do?

Answer 20

ensures initiation of transcription from specific sites, and reduces binding to non-specific sequences.

Question 21

what is the structure of the bacterial RNAP active site?

Answer 21

located at the base of the cleft formed by B and B’ jaws. contains 2 catalytic Mg2+ ions, one is tightly bound to the active site, and another arrives at the active site in complex with incoming NTP
there are three aspartate residues which are located on a short loop of the beta subunit, these coordinate the Mg2+