Sigma factors Flashcards
what is a sigma factor
(specificity factor) is a bacterial transcription initiation factor that enables specific binding of RNA polymerase to gene promoters.
sigma factor 70 is the
‘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
what are the key players in transcription
Core RNA polymerase, Sigma factor and a Promoter
in bacteria, a single
RNA polymerase enzyme is responsible for making all types of RNA
structure of bacterial RNA pol
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.
the core RNA pol cannot
initiate transcription by itself
requires sigma factors to recognise the promotor
what do sigma factors recognise
consensus sequences at promotor regions
where do sigma factors bind to DNA
-10 and -35 regions
Different sigma factors recognise different
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)
Differential recruitment of σ factors is central to
numerous stress responses in bacteria that promote growth & survival in adverse conditions
Sig factors could be a potential
drug target - they are unique to bacteria
expression of gene families can be controlled by regulating the availability of the
corresponding sigma factor
how are sigma factors regulated (3)
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
RpoS (σ38) is
the master regulator of general stress response
regulates approx. 10% of the bacterial genome
RpoS translation is increased by
multiple stimuli including: high cell density low temperature high osmolarity acidic pH
these conditions also REPRESS the normally rapid proteolysis of RpoS
high osmolarity, acidic pH, carbon starvation and high temperature all act to
boost the levels of RpoS (σ38) and thus promote the activation of RpoS-dependent genes in the stress response
PHOSPHORYLATED RssB directs RpoS for degradation to
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
IraP, IraM & IraD are
anti-adaptor proteins that inhibit RssB, stabilizing RpoS
along with suppression of ArcB, P, M & D increase levels of RpoS
IraP is activated by
the stringent response (pppGpp)
IraM is activated by
magnesium starvation AND PhoP/Q
ArcB monitors the cellular energy state so
during energy starvation the phosphorylation of RssB by ArcB is reduced, leading to reduced proteolysis of RpoS.
Bacterial stress responses are controlled by
PhoP/Q and RpoS
What is the role of RpoE?
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)
RpoE activation results in
Periplasmic folding machinery Proteases (to break down misfolded proteins) Lipid A biosynthesis Lipoproteins Proteins with periplasmic functions
RpoE senses stress in the cell envelope by
using ANTI SIGMA factor RseA
it is complexed to RpoE to suppress its activation
RseB further stabilises RseA
Misfolded proteins in the PERIPLASM is a marker of envelope stress, and will activate the RpoE pathway by
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
Activated DegS cleaves
RseA in the periplasm
Activated YaeL cleaves
RseB in the cytoplasm which releases RpoE
Once RpoE is free in the cytoplasm it complexes with
RNA pol and activates envelope stress genes
Misfolded proteins in the CYTOPLASM is a marker of heat shock and will activate
the ‘heat shock response’
includes molecular chaperones to try to help refold proteins
and proteases to remove badly damaged proteins
Molecular chaperones of the heat shock response
DnaK-DnaJ-GrpE
GroEL-GroES (major)
Proteases of the heat shock response
ClpXP
Lon, FtsH
Structure of ClpXP protease
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).
Structure of GroE chaperone
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
RpoH activates the
Heat shock response (sigma 32)
During the heat shock response, RpoH levels rise by
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)
sigma factors can be a
novel target for antimicrobials - they are unique to bacteria and govern many stress responses and virulence factors
methods of targeting sigma factors (3)
- blocking RNA pol - SI24 cyclic peptide inhibitor targets RpoE BUT in vivo it did not work because it could not cross the cell envelope
- 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)
- 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)
Summary of sigma factors
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
RpoS associated proteins
RssB, ArcB, ClpXp, IraP, IraM & IraD, pppGpp
RpoE associated proteins
RseA, RseB, DegS and YaeL
RpoH associated proteins
DnaK-DnaJ-GrpE, GroEL-GroES, ClpXP, Lon, FtsH
