Transcription 2 Flashcards
What are the similarities between replication and transcription?
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.
What are the differences between replication and transcription?
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.
How many RNA polymerases are there is prokaryotes?
1
How many RNA polymerases are there in eukaryotes?
3
Is the 3D structure of RNA polymerase similar between eukaryotes and prokaryotes?
Yes, the 3D structure is highly conserved between prokaryotes and eukaryotes.
What does the fact that the 3D structure of RNA polymerase is very similar between eukaryotes and prokaryotes suggest?
Implies that they have an identical catalytic mechanism.
How does the addition of new nucleotides to the polynucleotide chain occur?
By nucleophilic attack.
What does RNA polymerase require for activity?
DNA. Ribonucleotides and Mg^2+.
What does RNA polymerase use Mg^2+ to do?
Mg^2+ chelates two phosphate groups and this orientates the nucleotides in the active site ready for polymerisation.
What is the size of RNA polymerase in E.coli?
Very large - 400kD.
What are the kinds of subunits in RNA polymerase in E.coli?
alpha, beta, beta’, omega, sigma.
Which of the subunits make up the holoenzyme?
alpha x2, beta, beta’, omega, sigma.
Which of the subunits of the holoenzyme is a coenzyme?
sigma.
What is the function of the sigma subunit?
Sigma helps enzyme to recognize specific DNA sequences called promoter, initiate transcription, then dissociates.
What is the subunit composition of the core enzyme (apoenzyme) and what is its function?
alpha x2, beta, beta’, omega.It is involved in carring out catalysis / chain elongation
What is the key step in transcription?
The decision to express a gene. (Initiation)
Where does the holoenzyme bind before transcription?
Holoenzyme binds to about 70bp before transcription start site.
How can DNA protein binding be determined?
By DNA footprinting experiments - investigating the sequence specificity of DNA-binding proteins in vitro.
Where do critical conserved sequences occur in DNA?
They occur at -10 and -35 regions from the transcription start site - where sigma binds.
RNA polymerase recognises typical E.coli promoters, which sigma does the RNA polymerase contain?
Sigma 70
Promoter sequences vary, however where are they most likely to show similarity?
In the -10 and -35 regions.
Where would you find UP (upstream) elements and what do they do?
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.
What is the role of sigma in initiation?
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).
How does sigma allow for specific, regulated gene expression?
Different sigma factors permit binding to different promoters.
Which structure of the sigma factor helps it recognise DNA sequences and bind?
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).
What is the role of the alpha subunit?
Binds regulatory sequences/proteins.
What is the role of the beta subunit?
Forms phosphodiester bonds.
What is the role of the beta’ subunit?
Binds DNA template.
What is the role of the sigma subunit?
Promoter recognition.
What is the role of the omega subunit?
RNAP assembly.
What happens when the sigma subunit recognises the promoter region? (Initiation - 1)
It causes a conformation change in the RNA polymerase to form a closed complex with the DNA intact (not unwound).
What happens after the RNA polymerase has formed the closed complex? (Initiation -2)
Isomerisation of the closed complex occurs to form the open complex with DNA partially unwound around -3/-15.
After the open complex has formed and DNA has unwound what occurs? (Initiation -3)
The active site of RNA polymerase is exposed at the transcription initiation site and the first ribonucleotide is added.
What is promoter clearance? (Initiation - 4)
This means that the sigma factor dissociates as its affinity for the promoter is lost, leaving the core enzyme ready for elongation.
Describe initiation.
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.
