Sigma Factors

Question 1

What is transcriptional control and why is it important?

Answer 1

Control of gene regulation. DNA to RNA to protein.
Environmental change turns genes on and off, which leads to the production of proteins to deal with new environments.
Important so you don’t waste energy, display genes at the right time, avoid exposure to eg. pathogens.

Question 2

What are the different stages of regulation?

Answer 2

Transcription- initiation, elongation, termination.
Processing- turnover.
Translation.
Protein processing.

Question 3

What are the types of regulation?

Answer 3

Sigma factors.
Second messenger molecules- PPGPP, cDiAmp in prokaryotes.
Changes in number of transcripts- could go up or down.
Small regulatory RNAs and riboswitches- PrFA (transcription factor), stem loop structure- hides SD (ribosome binding site) seq. (at 30o).
DNA rearrangements.
At 37- Melts, RBS open, genes open for expression.
All of these need to be controlled to ensure the right response at the right time.

Question 4

What are the main genetic parts?

Answer 4

Single gene-promoter (binds RNAP) and operator (binds regulatory protein).
Operon- Operon- multiple genes in one transcript.
Regulon.
Stimulon.
Transcription generally happens in an independent direction to translation.
+1 promoter- where transcript starts.
Translated seq can be shorter than transcribed seq.

Question 5

What is an operon?

Answer 5

Functioning unit of DNA where mu.ltiple genes are controlled by a single promoter

Question 6

What is a regulon?

Give an example of a sigma factor found in the E.coli RpoS regulon.

Answer 6

Collection of genes or operons under regulation by the same regulatory protein.
RpoS (sigma-S, sigma-38)- starvation sigma factor. Can bind to various operons to help survival in stressful environments.

Question 7

What is the structure of RNA?

Answer 7

Contain ribose instead of deoxyribose.
Bases are A,G,C,U.
Uracil pairs with adenine.
Small chemical difference from DNA, but large structural differences.
Single stranded helix.
Ability to fold into 3D shapes - can be functional.

Question 8

What are the types of RNA?

Answer 8

Messenger RNA (mRNA)
Ribosomal RNA (rRNA)
Transfer RNA (tRNA)
Small regulatory RNA

Question 9

What structures can RNA have?

Answer 9

Various structures.
Secondary structure- stem loop, hairpin.
Tertiary- folding into a pseudoknot.
Function a bit more like protein than DNA- folding structures can give different domains.

Question 10

What are the stages of RNA synthesis?

Answer 10

Binding of RNA polymerase.
Separation of DNA.
Base pairing of nucleoside triphosphate to starting base in DNA.
Nucleosides added 5’ to 3’ through formation of phosphodiester bonds.

Question 11

What is RNA polymerase?

Answer 11

A large molecular machine (4 proteins in one complex: alpha 2x, beta, beta prime, omega).
Binds DNA and reads sequences. Polymerises RNA.
(omega- function unknown, in vitro- can reformat denatured RNA into functional form).

Question 12

What is the structure of a promoter?

Answer 12

Upstream element (-70): UP elements/alpha-binding. Bent DNA. Binding of activators.
Core promoter: has -35, spacer, then -10. Sigma recognition. Bindings of activators and repressors.
There are consensus sequences in the -35 and -10 regions of the core promoter. They have difefrent consensus sequences for different sigma factors.
Downstream element (+1):
promoter clearance. Binding of repressors.

(+1 in translation DIFFERENT to ATG for translation.
UTR= untranslated region.
-10 and -35 boxes are vital for binding sigma factors. (eg. -10 is 10 back from +1))

Question 13

What is sigma factor and what does it do?

Answer 13

Sigma factor associates with RNA polymerase (holoenzyme- core RNAp + sigma factors).
Complete RNAp scans to find -10/-35, binds, forms closed complex.
One sigma factor guides complex to site.
Disassociation of sigma factor makes the open complex, transcription bubble, RNAp can function.
Can achieve specificity of transcription by using different sigma factors.
(Some antibiotics work at this stage (rifamycin- blocks open complex formation. Used to treat eg. tuberculosis, leprosy etc.)

Question 14

How do proteins bind to DNA?

Answer 14

Non-specific binding is primarily electrostatic with some shape recognition.

Specific binding involves direct interaction between the nucleic acid and protein- Main two main categories are base readout and shape readout.

Proton donors and acceptors.
Van der Waal interactions.
Major versus minor grooves.
Nucleic acid “bendability” and flexibility.
Water molecules and cations.

Meeting of right bonds for specific interaction of RNAp and DNA.

Question 15

How do proton donors and acceptors work in the major and minor grooves of DNA?

Answer 15

All four bases can be distinguished in the major groove.
Can only distinguish A-T, T-A from C-G, G-C in minor groove.
The proton donors and acceptors on bases and phosphates (and the 2’OH of ribose) can form specific H-bonds with amino acids of proteins.

Question 16

How is specificity achieved in response to different signals? Give examples of different sigma factors.

Answer 16

Multiple sigma factors
- s70 
Guides RNA polymerase to most genes.
 - s32 
Active when cell is stressed by heat.
Heat-shock response.
At normal temps, it makes use of folded mRNA, not translated much. Heat shock proteins stay folded.
-Bs s32 
Active when cell is stressed by heat.
High temps, mRNA unfolded, 32 made, bind and degrade heat shock proteins. Prevents toxicity.
32 works with RNAp.

Question 17

What are differences and similarities in different sigma factors?

Answer 17

They all maintain a similar fold, but may have different sequences.

Question 18

How do you make a Sigma factor function only when you need it?

Answer 18

Can be controlled at multiple levels of production.
Using an anti-sigma factor (and an anti-anti-sigma factor etc!).
Spatial separation e.g. endospore formation in
Bacillus subtilis.
Or using combinations of all of the above.

Extracytoplasmic sigma factor- responds to extracellular signal- part of factor is cleaved off which activates factor.
Can have a heirarchal transcription route (one sigma factor activates transcription of the next etc…)

Question 19

What are the stages in the Bacillus subtilis sporulation cycle?

Answer 19

Germination, vegetative state, starvation state, onset state, commitment state, engulfment state, maturation state, mother cell lysis, and so on.

Question 20

How is sporulation activated? What are the first steps?

Answer 20

Vegetative cell- sigmaF is inactive.
Sporulation activated by Spo0A- phosphorylated in startvation- activates sporulation.
First- spollAA, spollAB and s-F genes transcribed
spollAB= anti-s-F.
If unbound, F is free.
AB can be removed by spollAA (anti-anti-s-F)- spollAB degraded.
Always making AB- F usually tied up.
SpollAB and s-F is a specific protein protein interaction.

Question 21

What happens to sigma F in the forespore?

Answer 21

Sigma F is active.

Question 22

What are the stages in the forespore

Answer 22

Sigma F is active.
Spatial separation occurs.
Second chromosome gets pumped into the spore so that ori goes first (genes go through in a very set order).
SpollAB almost at opposite end of chromosome.
Normal route (as before) in mother cell.
In forespore- F bound to AB, AA degrades AB.
Free F not bound again- F active, because SpollAB gene isn’t in the spore yet. Time limited period.

Question 23

How was the link between genetic asymmetry and protein asymmetry tested?

Answer 23

Test: REVERSE genetic asymmetry- Put anti-F coding region into forespore, and see that more protein happens.
Delete gene, put it back in near ori in forespore.
Protein in forespore would inhibit sporulation- s-F held tight due to transcription of anti-F.

Question 24

How is sporulation controlled in Bacillus subtilis?

Answer 24

Sporulation in Bacillus subtilis is controlled by spatial separation of the sigma factor from the anti-sigma factor.

Breaking the spatial separation inhibits sporulation.