Transcription mechanism + Regulation of initiation

Question 1

Which organisms have timing and location of transcription and translation having an overlap?

Answer 1

Bacteria. - eukaryotes have them separated

Question 2

What are the requirements for transcription?

Answer 2

1. Single stranded (ss) DNA template. - non-coding DNA strand acts as template.
2. All 4 RNA triphosphate nucleotides.
3. DNA dependent RNA polymerase (a holoenzyme consisting of sub units).

Question 3

What are the different forms of RNA polymerase?

Answer 3

Core enzyme
Holoenzyme

Question 4

What does the core enzyme do?

Answer 4

Catalysing DNA-directed RNA synthesis.

Question 5

What are the different parts of a core enzyme?

Answer 5

α2ββ’ (ω)

α - 40 kD each: function in enzyme assembly and activation.
β - 155 kD: catalytic domain and nucleotide binding.
β’ - 160 kD: template binding
ω - 6 kD; function unknown.

Question 6

What does the holoenzyme do?

Answer 6

Capable of catalysing DNA-directed RNA synthesis and initiating RNA synthesis at the correct locations.

Question 7

What are the different parts of the holoenzyme?

Answer 7

α2ββ’σ

α - 40 kD each: function in enzyme assembly and activation.
β - 155 kD: catalytic domain and nucleotide binding.
β’ - 160 kD: template binding
σ - various sizes; specificity factor, promoter binding and open complex formation.

Question 8

What direction does the coding strand go?

Answer 8

5’ to 3’

Question 9

What direction does the template/non-coding strand travel in relative to the coding strand?

Answer 9

3’ to 5’

Question 10

Which direction is upstream of the transcription initiation site (relative to coding strand (strand on top))?

Answer 10

towards the 5’.

Question 11

What are the stages of transcription?

Answer 11

1. Template recognition.
2. Initiation.
3. Elongation.
4. Termination.

1&2 are a part of initiation

Question 12

What happens in template recognition?

Answer 12

RNA polymerase binds to duplex DNA. Then DNA is unwound at promoter. If there is a sigma factor then a holoenzyme was used.

Question 13

What happens in the initiation part of transcription?

Answer 13

Chains of 2-9 bases are synthesised and released.

Question 14

What happens in the elongation stage of transcription cycle?

Answer 14

RNA polymerase moves; RNA is synthesised by base pairing with one strand of DNA (sigma is released). Unwound region moves with RNA polymerase. RNA polymerase reaches end of gene.

Question 15

What happens in the termination part of transcription?

Answer 15

RNA polymerase and RNA are released at terminator and DNA duplex reforms.

Question 16

What is the K\/I equation for the equilibrium equation?

Answer 16

Ki = RPc/(R+P)
R = RNA polymerase
P = promoter
RPc = closed promoter complex

Question 17

What are the kinetics of transcription initiation?

Answer 17

R + P –Ki–> <–K-i– RPc –K2–> <–K-2– RPo –K3–> <–K-3– RPi –K4–> RPE

i = one or initiation
o = open
c = closed
E = elongation
From initation to elongation there is no longer a reversible reaction
2 = II
3= III
4 = IV

Question 18

What is the rate of open complex formation called?

Answer 18

KII or K2

Question 19

What happens in K3?

Answer 19

There is NTPs in the forward reactions.
If the backward reaction happen (goes from initiation to open promoter complex) then abortive transcripts happened.

Question 20

What bit of kinetics transcription initiation is called the promoter clearance?

Answer 20

KIV or K4
Its the bit that is irreversible when it moves from initiation to elongation.

Question 21

What happens when polymerase binds to DNA?

Answer 21

It binds non-specifically to DNA with low affinity and migrates, looking for promoter. The sigma subunit recognises promoter sequence.
RNA polymerase holoenzyme and promoter form closed promoter complex (DNA not unwound) - Ki = 10E-6 to 10E-9 M.
Polymerase unwinds about 12 pairs to form open promoter complex - Kii =10E-14M

Question 22

What are promoters?

Answer 22

Sequences in the DNA just upstream of transcripts (coding sequences) that define the sites of initiation.

Question 23

What is the role of the promoter?

Answer 23

Attract RNA polymerase to the correct start site so transcription can be initiated.

Question 24

How were promoter sites defined (bacteria)?

Answer 24

1. Bioinformatics - comparison of known start sites to identify consensus sequences - not wholly conserved but still shows high conservation.
   Regions of similarity found between -10 and -35 bases
2. Biochemical - DNAse protection - DNAse foot printing can be used to identify sites where RNA polymerase is in close contact with DNA.
3. Genetic - mutations of the sites can lead to elimination/reduction of transcriptional initiation at a promoter. Strong promoters have sites that are very similar to the consensus sequence while weak promoters show many differences.

Question 25

What happens in DNAse protection?

Answer 25

1. Add purified RNA polymerase to DNA.
2. Digest extensively with DNAse (digests all uncovered DNA into nucleotides).
3. Isolate and identify the protected region (usually about 60 bp in prokaryotes.

Question 26

What do sigma factors do?

Answer 26

1. increase initiation rate (not elongation rate).
2. Contacts with core occur throughout its length.
3. Can no bind DNA unless bound to core (must bury inhibitory region).
4. Decreases affinity of core for nonspecific DNA.
5. Recognises bases in coding strand at -10 and also double strands at -35.

Question 27

What is the function of sigma factor subunit?

Answer 27

Subunit of RNA polymerase - initiation factor.
Ensures RNA polymerase binds stably to DNA promoter.
Destabilises nonspecific binding to non-promoter DNA.
Sigma stabilises specific binding to promoter DNA. (Affinity increases in holoenzyme in specific promoter DNA).
Accelerates the search for promoter DNA.
Diff sigma factors recognise diff promotes and have diff regulons.

Question 28

What is the principal sigma factor?

Answer 28

RpoD - cannot be knocked out as it is very important. The other sigma factors have specialised functions eg work when heat shocked.

Question 29

When does sigma 54 work?

Answer 29

Involved in activation of genes for nitrogen starvation conditions.
Can bind to promoter without the core RNA polymerase (it has no inhibitory domain) - more like eukaryotic transcription factor.
Can only facilitate closed complex formation - specific activators needed for open complex formation and successful transcriptional initiation.

Question 30

What is the interaction between DNA and RNA polymerase?

Answer 30

1. Core RNA polymerase is stored at non-specific DNA sites before sigma binds. Core has high affinity for DNA, which is increased by the presence of nascent RNA. Affinity for loose binding sites too high to find promoters efficiently from other sequences.
2. By reducing the stability of the loose complexes, sigma allows the process to occur much more rapidly and by stabilising the association at tight binding sites, the factor drives the reaction irreversibly into the formation of open complexes.
   3) When sigma released, the core plus nascent transcript (ternary complex) is essentially locked onto the DNA until termination.

Question 31

What happens in abortive initiation?

Answer 31

AKA cycling.
RNA pol transcribe 2-9 nt and then restarts.
Does not leave the promoter.
May occur several-hundreds times before elongation.
Only 8 bases can fit in active site of an enzyme. Hence the abortive products of 8-10 base.

Question 32

How many bases can fit in an enzymes active site?

Answer 32

8

Question 33

What happens in the sigma cycle?

Answer 33

Closed promoter complex (moderately stable).
Sigma subunit binds to -10 region.
Holoenzyme + sigma factor.
Open promoter complex (highly stable).
Holoenzyme has high affinity for promoter regions due to sigma.
Once initiation takes place, RNA polymerase does not need very high affinity for the promoter.
Sigma factor dissociates from core after few elongation reactions.
Elongation takes place w/ core.
Sigma rebinds to other core enzymes.

Question 34

What happens in elongation?

Answer 34

1. After 8-9 bp RNA synthesis occurs, sigma released and recycled in other reactions.
2. RNA polymerase completes the transcription at 30-50 nt/sec.
3. DNA untwists rapidly and reanneals behind enzyme.
4. Part of new RNA strand is hybrid DNA-RNA, but most RNA is displaced as helix reforms.

Question 35

Why is coding strand called that?

Answer 35

Same as mRNA but uses uracil. Template strand is used for mRNA to code off of.

Question 36

What causes supercoiling during transcription?

Answer 36

DNA in front of polymerase is overwound and DNA behind in underwound.
Transcription causes stress.

Question 37

What needs to be done to compensate for supercoiling during transcription?

Answer 37

Two enzymes are needed: gyrase in front to introduce negative supercoils and topoisomerase trailing to relax the supercoils.

Question 38

What are the two types of termination?

Answer 38

Factor independent
Factor dependent (Rho dependent termination)

Question 39

What does factor independent termination need?

Answer 39

Inverted repeats (GC rich region - very stable)
Followed by run of Ts on coding strand.
Forms hairpin loop.
RNA:RNA hybrid very stable.
Transcription bubble is destabilised RNA polymerase falls off.

Question 40

What does factor dependent termination need?

Answer 40

Requires proteins that interact w/ polymerase or transcript to stop transcription.
E coli has 3 termination factors: Rho (best known), Tau and NusA.

Question 41

What happens in Rho-dependent termination?

Answer 41

Works only if RNA not being translated. -Bacteria can have simultaneous transcription/translation.
Rho is RNA-dependent ATPase only cleaves ATP if RNA is present.
Rho is RNA-DNA helicase. Will only unwind double helix if RNA:DNA hybrid.
1. Termination can not occur if rut (Rho Utilisation site) is being translated.
2. Rho binds to rut mRNA and slides along mRNA chasing polymerase.
3. RNAP pauses at termination site; Rho catches polymerase. (Rho sliding requires hydrolysis of ATP).
4. Helicase activity unwinds RNA:DNA hybrid.
5. Disruption of RNA:DNA hybrid causes RNA poly and mRNA to fall off.