Sigma factors

Question 1

what is a sigma factor

Answer 1

(specificity factor) is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.

Question 2

sigma factor 70 is the

Answer 2

‘housekeeping’ sigma factor
stresses cause the alteration of utilisation of sigma factors (e.g 54) brings about global change in gene expression that allows adaptation

Question 3

what are the key players in transcription

Answer 3

Core RNA polymerase, Sigma factor and a Promoter

Question 4

in bacteria, a single

Answer 4

RNA polymerase enzyme is responsible for making all types of RNA

Question 5

structure of bacterial RNA pol

Answer 5

alpha-2-beta-beta-prime

identical alpha subunits – alpha 1 and 2 are encoded by the rpoA gene. The alpha subunits form the core enzyme, recognise the DNA promoter regions and aid interactions with TFs

the beta and beta-prime subunits are encoded by the rpoB and rpoC genes respectively, which are actually the catalytic centres of the Rpol, responsible for the synthesis of the RNA

The omega subunit aids the proper folding and recruitment of the beta-prime subunit to the core RNA polymerase.

Question 6

the core RNA pol cannot

Answer 6

initiate transcription by itself

requires sigma factors to recognise the promotor

Question 7

what do sigma factors recognise

Answer 7

consensus sequences at promotor regions

Question 8

where do sigma factors bind to DNA

Answer 8

-10 and -35 regions

Question 9

Different sigma factors recognise different

Answer 9

recognition sequences

e.g. sig54, nitrogen assimilation genes
sig32, heat shock response
e.g. sig70RpoD, -35, major sigma factor for normal growth (regulates 1000 genes)

Question 10

Differential recruitment of σ factors is central to

Answer 10

numerous stress responses in bacteria that promote growth & survival in adverse conditions

Question 11

Sig factors could be a potential

Answer 11

drug target - they are unique to bacteria

Question 12

expression of gene families can be controlled by regulating the availability of the

Answer 12

corresponding sigma factor

Question 13

how are sigma factors regulated (3)

Answer 13

Changing the rate of synthesis of the sigma factors

Changing the rate of degradation of the sigma factors Through the activity of anti-sigma factors

Question 14

RpoS (σ38) is

Answer 14

the master regulator of general stress response

regulates approx. 10% of the bacterial genome

Question 15

RpoS translation is increased by

Answer 15

multiple stimuli including:
high cell density
low temperature
high osmolarity
acidic pH

these conditions also REPRESS the normally rapid proteolysis of RpoS

Question 16

high osmolarity, acidic pH, carbon starvation and high temperature all act to

Answer 16

boost the levels of RpoS (σ38) and thus promote the activation of RpoS-dependent genes in the stress response

Question 17

PHOSPHORYLATED RssB directs RpoS for degradation to

Answer 17

ClpXp protease

RssB needs to be phosphorylated to do this
it is phosphorylated by ArcB
under starvation conditions, ArcB will be suppressed so RpoS levels in the cell increase

Question 18

IraP, IraM & IraD are

Answer 18

anti-adaptor proteins that inhibit RssB, stabilizing RpoS

along with suppression of ArcB, P, M & D increase levels of RpoS

Question 19

IraP is activated by

Answer 19

the stringent response (pppGpp)

Question 20

IraM is activated by

Answer 20

magnesium starvation AND PhoP/Q

Question 21

ArcB monitors the cellular energy state so

Answer 21

during energy starvation the phosphorylation of RssB by ArcB is reduced, leading to reduced proteolysis of RpoS.

Question 22

Bacterial stress responses are controlled by

Answer 22

PhoP/Q and RpoS

Question 23

What is the role of RpoE?

Answer 23

maintaining the cell envelope
sensory link between the cell membrane and periplasm to the inside of the cell
sigma factor E, activated by stress to the envelope (e.g. ABs, heat)

Question 24

RpoE activation results in

Answer 24

Periplasmic folding machinery 
Proteases (to break down misfolded proteins)
Lipid A biosynthesis 
Lipoproteins 
Proteins with periplasmic functions

Question 25

RpoE senses stress in the cell envelope by

Answer 25

using ANTI SIGMA factor RseA it is complexed to RpoE to suppress its activation RseB further stabilises RseA

Question 26

Misfolded proteins in the PERIPLASM is a marker of envelope stress, and will activate the RpoE pathway by

Answer 26

binding to the PDZ domain of DegS and YaeL in the periplasm DegS is then activated as a protease and cleaves RseA in the periplasm YaeL cleaves RseB

Question 27

Activated DegS cleaves

Answer 27

RseA in the periplasm

Question 28

Activated YaeL cleaves

Answer 28

RseB in the cytoplasm which releases RpoE

Question 29

Once RpoE is free in the cytoplasm it complexes with

Answer 29

RNA pol and activates envelope stress genes

Question 30

Misfolded proteins in the CYTOPLASM is a marker of heat shock and will activate

Answer 30

the 'heat shock response' includes molecular chaperones to try to help refold proteins and proteases to remove badly damaged proteins

Question 31

Molecular chaperones of the heat shock response

Answer 31

DnaK-DnaJ-GrpE | GroEL-GroES (major)

Question 32

Proteases of the heat shock response

Answer 32

ClpXP | Lon, FtsH

Question 33

Structure of ClpXP protease

Answer 33

ClpX is an atp dependent chaperone that puts the protein into: ClpP is a peptidase The ATPase provides the energy for protein unfolding and translocation into the ClpP protease complex other proteases that combine the ‘unfoldase’ and protease activities within a single protein (e.g. Lon and FtsH proteases).

Question 34

Structure of GroE chaperone

Answer 34

GroEL has 14 identical subunits (2 rings of 7) Forms a highly hydrophilic chamber GroES forms a cap on the chamber, creating an enclosed space in a normally folded protein, hydrophobic parts will be inside the protein in a misfolded protein, some of these residues will be on the outside - they bind to GroEL and the confined hydrophilic space of the chamber forces correct folding using ATP

Question 35

RpoH activates the

Answer 35

Heat shock response (sigma 32)

Question 36

During the heat shock response, RpoH levels rise by

Answer 36

increase dramatically and rapidly by: increasing rate of translation (mRNA has a different secondary structure at higher temps) decreasing rate of degradation (RpoH normally is bound to DnaK for degdradation BUT during heat shock it will bind preferentially to misfolded proteins, releasing RpoH to activate the heat shock genes)

Question 37

sigma factors can be a

Answer 37

novel target for antimicrobials - they are unique to bacteria and govern many stress responses and virulence factors

Question 38

methods of targeting sigma factors (3)

Answer 38

1. blocking RNA pol - SI24 cyclic peptide inhibitor targets RpoE BUT in vivo it did not work because it could not cross the cell envelope 2. RpoN (sig 54) 'molecular roadblock' - peptide that blocks the RpoN consensus sequence in the promotor so RNA pol can't bind but still doesn't work irl as it can't get through the envelope Plasmid expressing RpoN* (mutant roadblock) was transformed into Pseudomonas aeruginosa Resulted in differential expression of approx. 700 genes Reduced activity of numerous virulence determinants including Swimming motility, Elastase and pyocyanin, Pyoverdine (siderophore) 3. Preventing dissociation from anti-sigma factor - FPSS as a sigma-B inhibitor of Listeria and Bacillus, better approach as responded to external treatment (unlike 1 and 2)

Question 39

Summary of sigma factors

Answer 39

σ70 is housekeeping/normal growth RpoS (σ38) is the master regulator of stress response RpoE (σE) regulates the envelope stress response RpoH (σ32) regulates the heat shock response

Question 40

RpoS associated proteins

Answer 40

RssB, ArcB, ClpXp, IraP, IraM & IraD, pppGpp

Question 41

RpoE associated proteins

Answer 41

RseA, RseB, DegS and YaeL

Question 42

RpoH associated proteins

Answer 42

DnaK-DnaJ-GrpE, GroEL-GroES, ClpXP, Lon, FtsH