Prokaryotic gene expression L10-14

Question 1

In a bacteria what is the typical half life of mRNA?

Answer 1

few minutes
mRNA is continually being made and then degraded
Proteins have half lives yet these are longer

Question 2

What are the stop codons in bacteria?

Answer 2

UAA UGA UAG

Question 3

Is there a delay b/w transcription and translation in Proks?

Answer 3

No since there is no nucleus in Proks so no need for export b/w compartments
As soon as the shine dalgarno seq becomes exposed out of RNAP, ribosomes can bind and begin translation.
Rate of translation is almost the same as the av. rate of transcription
Rate of RNAP = 40-100NTs per second (slower than DNA replication)

Question 4

What is the rate of ribsomes in Proks?

Answer 4

33 amino acids per second

Question 5

Differences b/w Pro and Euk

Answer 5

Proks-respond efficiently at the transcription level
Euks-immediate responses to starvation tend to be post-translational modifications of proteins since can’t instantly produce proteins like bacteria, so proteins are made in case they are needed. Switches on/off at the level of protein
Proks-one form of RNAP-transcribes all the genes (ribosomal/tRNA genes and protein genes)
Euks-5 forms of RNAP
Proks-have sigma factor for recognising promoter
Euks-Have TATA binding protein

Question 6

What is the E.coli RNAP composed of?

About 2000 RNAPs exist in each cell

Answer 6

alpha - core assembly, DNA binding, TF interactions
beta/beta’ - catalytic site
sigma70 - promoter recognition
omega - chaperone activity (beta’ associated)

When RNA synthesis occurs 2 Magnesiums are present to catalyse the phosphodiester bond formation

Question 7

How many NTs after transcription initiation till the sigma factor dissociates?

Answer 7

15/20 NTs of RNA (conf of RNAP changes) As RNA becomes excluded it pushes off the sigma subunit

Question 8

What is the promoter seq for RNAP?

NB/ the core promoter is only in the middle of the 70bp footprint of RNAP (-50 to +20)

Answer 8

TTGACA(-35) 17bps TATAAT(-10) 5-7bps A
tiny tim gave anna chlamydia and then anna thought all about tim
Promoters are conserved in both their sequence and their SPACING

Question 9

How many promoters contain an UP element?

Answer 9

1/4
Not recognised by sigma factor
alpha subunits bind there.
The alpha subunit has 2 domains and binding activity
N terminal domain, a linker and a C terminal domain
alpha domain binds to AT rich region upstream of -35

Question 10

What is the favoured base for initiating transcription?

Answer 10

A

Question 11

Sigma factor

Answer 11

r4 (binds to -35)
linker
r2 (binds to -10) often captures non-template strand during transcription
The linker winds its way through the middle of RNAP and this is the channel mRNA takes when the sigma factor becomes released
Differences in the -35 hexamer effects KD (increases)
Differences in the -10 region effects K2
-35 is responsible for binding
-10 is responsible for isomerisation
Strong UP element effects KD (decreases)

Question 12

How much RNAP is free in a cell out of 2000 molecules?

Answer 12

660
2/3rds are in middle of gene producing RNA
About 750 molecules of sigma70 per cell

Question 13

Concentrations of types of sigma factors bound to RNAP

Answer 13

75% sigma70 housekeeping
10% sigma54 N metabolism (-24/-12)
15% sigma28 flagella

Question 14

How many TFs do E.coli have?

Answer 14

132

Question 15

Activators/Repressors

Answer 15

Approx 70% of a sigma70 dependent promoters are regulated by one repressor
Approx 50% of a sigma70 dependent promoters are regulated by one activator
Many TFs are ‘global regulators’-they have a pleiotropic effect
In general each TF can only sense one thing about the environment
In bacteria repression is a more common strategy for regulation than Euks (intrinsic repression due to chromatin structure)

Question 16

What is the difference b/w lac repressor and trp repressor?

Answer 16

LAC
1000Xfold repression with auxiliary operators
tetramer (dimer of dimers)
only regulates 1 operon
TRP
70Xfold repression
dimer
regulates 3 operons (regulates its own synthesis)
–feedback inhibition loop, negative feedback, make too much and the trp repressor will decrease its synthesis
Trp repressor binds to DNA as a repressor-tryptophan complex, tryptophan is a corepressor not an inducer

Question 17

Where does CAP (camp activator protein) bind to on the lac operon?

Answer 17

-61 (-41/-71 etc) -41 is within the promoter and RNAP snuggles on the other side and makes 3 diff interactions with RNAP
doesn’t work if it is at -60/-62
Single NT specific
cap protein BENDS the DNA when it binds to it
C terminal domains of the alpha subunits of RNAP are held on to by cap

Question 18

sigma54

Answer 18

Transcriptional activators that activate sigma54 dependent promoters are different from CAP-normally higher order multimers e.g. hexamer/septamer that forms a ring
Also ATP hydrolysing enzymes
Promoters regulated by sigma54 require special ATP-utilising transcription activators in order to form open complexes.
Binding sites for these transcription factors can be hundreds of bps away from the promoter (The activator and the RNAP are brought into contact via DNA bending)

Question 19

What are the two ways of terminating transcription in prokaryotes?

Answer 19

Rho independent/intrinsic terminators - RNA hairpin followed by a run of Us
Rho dependent-Rho ATPase motor protein binds to a rut site in the mRNA and follows RNAP along mRNA until RNAP reaches a pause site. Rho protein catches up and crashes into RNAP releasing the mRNA. Motor is kept running and pulls the RNA out the back of the RNAP. All interactions of the RNAP and DNA are overcome by this motor burning ATP.

Question 20

How does anti terminators work?

Answer 20

Nut box transcribed by RNAP from DNA- N protein binds here to the Nut box on RNA and the RNAP along with another protein and forms an extended RNA exit channel of the RNAP. This disrupts the spacing between the formation of the hairpin and run of Us so that the next coding region is transcribed.

Question 21

What molecule is incorporated to switch off/on riboswitches?

Answer 21

SAM (methyl donor)

Riboswitches can also control translation

Question 22

How long is the leader peptide when using this to control expression of operons?
The ribosomes made the leader peptide at the same time as the RNA is being synthesised
We are able to monitor Trp concentration by saying how quickly can a ribosome find a Trp charged tRNA to translate these codons

Answer 22

14aa
Attenuation by leader peptide formation controls expression of at least 6 E.coli amino acid biosynthetic operons (e.g. Trp, His)
2 aa’s require the insertion of Trp
Trp present-ribosome forms a hairpin b/w 3/4=terminator
Trp absent-ribosome stalls at R1 allowing R2/3 to form hairpin-transcription of trp operon occurs

Question 23

How long is the footprint of a ribosome?

Answer 23

40NTs long

Question 24

What is used for controlling translation?

Answer 24

Translation repressor proteins-bind to ribosome binding site

RBS is as ssRNA so can form secondary structures - therefore riboswitches can also control the access of ribosomes to the RBS (in trans-same strand of RNA)

Small regulatory RNAs (in cis) ~100NTs bind to RBS complementary antisense RNA-binds and overlaps RBS
Can activate translation by binding to a hairpin if a hairpin has formed over the RBS-competes with hairpin formation