Transcription 2

Question 1

What are the similarities between replication and transcription?

Answer 1

Both involve the addition of nucleotides to 3’ end of growing chain. Polynucleotide growth is always in the 5’ to 3’ direction. Both use of a DNA template. Both include three phases: Initiation, Elongation and Termination.

Question 2

What are the differences between replication and transcription?

Answer 2

Transcription does not require a primer.Not all the DNA is transcribed, but all the DNA is replicated. Only one strand of a DNA template is transcribed by RNA polymerase.

Question 3

How many RNA polymerases are there is prokaryotes?

Answer 3

1

Question 4

How many RNA polymerases are there in eukaryotes?

Answer 4

3

Question 5

Is the 3D structure of RNA polymerase similar between eukaryotes and prokaryotes?

Answer 5

Yes, the 3D structure is highly conserved between prokaryotes and eukaryotes.

Question 6

What does the fact that the 3D structure of RNA polymerase is very similar between eukaryotes and prokaryotes suggest?

Answer 6

Implies that they have an identical catalytic mechanism.

Question 7

How does the addition of new nucleotides to the polynucleotide chain occur?

Answer 7

By nucleophilic attack.

Question 8

What does RNA polymerase require for activity?

Answer 8

DNA. Ribonucleotides and Mg^2+.

Question 9

What does RNA polymerase use Mg^2+ to do?

Answer 9

Mg^2+ chelates two phosphate groups and this orientates the nucleotides in the active site ready for polymerisation.

Question 10

What is the size of RNA polymerase in E.coli?

Answer 10

Very large - 400kD.

Question 11

What are the kinds of subunits in RNA polymerase in E.coli?

Answer 11

alpha, beta, beta’, omega, sigma.

Question 12

Which of the subunits make up the holoenzyme?

Answer 12

alpha x2, beta, beta’, omega, sigma.

Question 13

Which of the subunits of the holoenzyme is a coenzyme?

Answer 13

sigma.

Question 14

What is the function of the sigma subunit?

Answer 14

Sigma helps enzyme to recognize specific DNA sequences called promoter, initiate transcription, then dissociates.

Question 15

What is the subunit composition of the core enzyme (apoenzyme) and what is its function?

Answer 15

alpha x2, beta, beta’, omega.It is involved in carring out catalysis / chain elongation

Question 16

What is the key step in transcription?

Answer 16

The decision to express a gene. (Initiation)

Question 17

Where does the holoenzyme bind before transcription?

Answer 17

Holoenzyme binds to about 70bp before transcription start site.

Question 18

How can DNA protein binding be determined?

Answer 18

By DNA footprinting experiments - investigating the sequence specificity of DNA-binding proteins in vitro.

Question 19

Where do critical conserved sequences occur in DNA?

Answer 19

They occur at -10 and -35 regions from the transcription start site - where sigma binds.

Question 20

RNA polymerase recognises typical E.coli promoters, which sigma does the RNA polymerase contain?

Answer 20

Sigma 70

Question 21

Promoter sequences vary, however where are they most likely to show similarity?

Answer 21

In the -10 and -35 regions.

Question 22

Where would you find UP (upstream) elements and what do they do?

Answer 22

Not in all E.coli promoters - but if present they will be in the -40 and -60 region. They strongly stimulate transcription at the promoters that contain them.

Question 23

What is the role of sigma in initiation?

Answer 23

Decreases ability of core enzyme to bind DNA non-specifically (by about 10^4).Allows holoenzyme to bind promoters (at -10) Allows holoenzyme to migrate along the DNA until a promoter is encountered (random walk).

Question 24

How does sigma allow for specific, regulated gene expression?

Answer 24

Different sigma factors permit binding to different promoters.

Question 25

Which structure of the sigma factor helps it recognise DNA sequences and bind?

Answer 25

The alpha helix which is followed by a turn (helix-turn-helix) forms a finger like protrusion that interacts with the major groove of DNA (hydrogen bonds and Van der Waals interactions with exposed bases).

Question 26

What is the role of the alpha subunit?

Answer 26

Binds regulatory sequences/proteins.

Question 27

What is the role of the beta subunit?

Answer 27

Forms phosphodiester bonds.

Question 28

What is the role of the beta’ subunit?

Answer 28

Binds DNA template.

Question 29

What is the role of the sigma subunit?

Answer 29

Promoter recognition.

Question 30

What is the role of the omega subunit?

Answer 30

RNAP assembly.

Question 31

What happens when the sigma subunit recognises the promoter region? (Initiation - 1)

Answer 31

It causes a conformation change in the RNA polymerase to form a closed complex with the DNA intact (not unwound).

Question 32

What happens after the RNA polymerase has formed the closed complex? (Initiation -2)

Answer 32

Isomerisation of the closed complex occurs to form the open complex with DNA partially unwound around -3/-15.

Question 33

After the open complex has formed and DNA has unwound what occurs? (Initiation -3)

Answer 33

The active site of RNA polymerase is exposed at the transcription initiation site and the first ribonucleotide is added.

Question 34

What is promoter clearance? (Initiation - 4)

Answer 34

This means that the sigma factor dissociates as its affinity for the promoter is lost, leaving the core enzyme ready for elongation.

Question 35

Describe initiation.

Answer 35

The sigma subunit recognises the promoter causing a conformation change in the RNA polymerase to form a closed complex with the DNA intact (not unwound). somerisation of the closed complex occurs to form the open complex with DNA partially unwound around -3/-15.The active site of RNA polymerase is exposed at the transcription initiation site and the first ribonucleotide is added. Then promoter clearance occurs.