Transcription 1

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 are the secondary structures of RNA polymerase?

Answer 6

Alpha and beta sheets.

Question 7

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

Answer 7

Implies that they have an identical catalytic mechanism.

Question 8

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

Answer 8

By nucleophilic attack.

Question 9

What does RNA polymerase require for activity?

Answer 9

DNA.

Ribonucleotides and Mg^2+.

Question 10

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

Answer 10

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

Question 11

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

Answer 11

Very large - 400kD.

Question 12

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

Answer 12

alpha, beta, beta’, omega, sigma.

Question 13

Which of the subunits make up the holoenzyme?

Answer 13

alpha x2, beta, beta’, omega, sigma.

Question 14

Which of the subunits of the holoenzyme is a coenzyme?

Answer 14

sigma.

Question 15

What is the function of the sigma subunit?

Answer 15

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

Question 16

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

Answer 16

alpha x2, beta, beta’, omega.

It is involved in carring out catalysis / chain elongation

Question 17

What is the key step in transcription?

Answer 17

The decision to express a gene. (Initiation)

Question 18

Where does the holoenzyme bind before transcription?

Answer 18

Holoenzyme binds to about 70bp before transcription start site.

Question 19

How can DNA protein binding be determined?

Answer 19

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

Question 20

Where do critical conserved sequences occur in DNA?

Answer 20

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

Question 21

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

Answer 21

Sigma 70

Question 22

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

Answer 22

In the -10 and -35 regions.

Question 23

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

Answer 23

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 24

What is the role of sigma in initiation?

Answer 24

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 25

How does sigma allow for specific, regulated gene expression?

Answer 25

Different sigma factors permit binding to different promoters.

Question 26

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

Answer 26

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 27

What is the role of the alpha subunit?

Answer 27

Binds regulatory sequences/proteins.

Question 28

What is the role of the beta subunit?

Answer 28

Forms phosphodiester bonds.

Question 29

What is the role of the beta’ subunit?

Answer 29

Binds DNA template.

Question 30

What is the role of the sigma subunit?

Answer 30

Promoter recognition.

Question 31

What is the role of the omega subunit?

Answer 31

RNAP assembly.

Question 32

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

Answer 32

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

Question 33

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

Answer 33

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

Question 34

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

Answer 34

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

Question 35

What is promoter clearance? (Initiation - 4)

Answer 35

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

Question 36

Describe initiation.

Answer 36

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.