Transcript Lecture 10 Powerpoint Presentation

Scotty Merrell
Department of Microbiology and Immunology
B4140
[email protected]
Regulation of Gene Expression II
Regulation
gene expression
Regulation
of ofGene
Expression
RNA polymerase
Regulatory proteins aa-tRNAs
RNA polymerase
DNA
Promoter Attenuator
operator
Transcription
Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination
Regulatory proteins
Antisense RNAs
mRNA
Ribosome
binding
site
Translation
Stop signal
Translational control
Translation initiation:
positive/negative
Degradation
Protein
Post-translational control
(e.g., proteolysis)
Translational Control of gene expression:
The Ribosome binds the Shine-Dalgarno sequence
upstream of the AUG start codon
http://www.mag.keio.ac.jp/~rsaito/Research/Survey/trans_pro.html
Translational control can occur at the level of
RNA-RNA interaction
(Antisense RNA)
1. Negative control: translation initiation can be inhibited by
antisense RNA which pairs with 5’ mRNA and blocks SD
sequence.
2. Positive control: antisense RNA binds with 5’ untranslated
region preventing formation of a secondary structure that
blocks the Shine-Dalgarno sequence.
Where does antisense RNA come from?
A. Antisense RNA can be transcribed from the opposite strand
of the same DNA fragment of the gene, thus the antisense
RNA complements the sense RNA completely.
B. Antisense RNA can be transcribed from a different genetic
location from the sense RNA and usually is not
completely complimentary.
Inhibition of translation by antisense RNA:
Example: ompF in E. coli
P
micF
Activator
RNAP
P/O
ompF
OmpF is one of the two
major outer membrane
porin proteins, that permit
the passive diffusion of
small, hydrophilic
molecules into the
periplasm of E. coli.
Activator
RNAP
Antisense
RNA
Inhibition
OmpF
(a) No antisense RNA
SD
ompF mRNA
Why would OmpF
expression
be modulated by
environmental
signals?
Translation
(b) Antisense RNA present
SD
ompF mRNA
Antisense RNA
SD is blocked
No translation
Activation of translation by antisense RNA:
a-Toxin expression in S. aureus
agrC
Acti
hld
Acti
va
hla
P
D agrB P2 P3 rnaIII
t i on
agrA
RNAIII
vatio
Acti
a-Toxin is a pore
forming toxin,
and is one of the
most important
S. aureus
virulence factors.
n
vatio
n
a-Toxin
Activation
(a) No RNAIII
hla mRNA leader
5’-hla mRNA
SD site is blocked
No translation
SD
AUG
Why is a-toxin
expression
modulated by the
environment?
3’
(b) RNAIII present
3’
5’
3’-RNAIII
5’-hla mRNA
AU
SD
G
SD site is relieved
Translation
Take home message:
Production of a protein can be regulated
at the level of translation.
This can be positive or negative regulation
and occurs independent of transcription.
Post-Translational Control -- Proteolysis
Degradation of a regulatory protein leads
to changes in gene expression
Example: E. coli SOS repair pathway--
LexA -- transcriptional repressor, control the
expression of numerous genes involved in
DNA repair, such as recA, uvrA, uvrB, uvrC, etc.
Post-translational control: proteolysis
Uninduced state
To other genes controlled by LexA
LexA repressor
...
lexA
Operator
...
recA
...
uvrA
...
uvrB
We see low level expression of all genes due to repression
Post-translational control: proteolysis
Induced state
DNA damage
Activated RecA
protease
Photodimer
LexA repressor
Cleaved LexA repressor
RecA
...
lexA
Operator
...
recA
...
uvrA
...
uvrB
Repression is relieved---high level expression
Proteolysis is important for many diverse functions
Swarmer
Cell
Stalked
Cell
Predivisional
Cell
Schematic diagram of the C.crescentus cell cycle. SW, swarmer cell; ST, stalked cell;
PD, predivisional cell. The eukaryotic nomenclature has been adapted for the stages
of the cell cycle. G1 corresponds to the SW cell where initiation of DNA replication is
inhibited by the CtrA protein (Quon et al., 1998). The S phase includes the ST cell,
where DNA replication is initiated, and the early PD cell. G2 corresponds to the late
PD cell, where the newly replicated chromosomes are segregated to the cell poles
and cell division gives rise to two daughter cells.
CtrA
ClpP mutant
ClpX mutant
CtrA is a major
regulator
in Caulobacter.
ClpP mutant
ClpX mutant
CtrA is a major
regulator
in Caulobacter.
CtrA degradation is
irregular in ClpP and
ClpX mutants
The cell-cycle is altered as a result
WT
Clp
mutant
Mixed
Population
G1
G2
Rifampicin
treated
Does anyone remember what the target
or mechanism of action is of rifampicin (rifampin)?
Does anyone remember what the target
or mechanism of action is of rifampicin (rifampin)?
Targets the DNA-dependent RNA polymerase-Inhibits RNA synthesis.
ClpP and ClpX are constitutively expressed
What could this mean in terms of the CtrA regulation we observed?
Model for C.crescentus cell-cycle control by the ClpXP protease. ClpXP is required for degradation of CtrA during the G1-to-S
transition and presumably also for removal of CtrA from the ST compartment of the late PD cell (G2). The phosphorylated
form of CtrA blocks replication in the SW cell and in the SW compartment of the late PD cell (OFF) (Quon et al., 1998).
Degradation and dephosphorylation of CtrA result in the onset of DNA replication in the ST cell and in the ST compartment of
the late PD cell (ON) (Domian et al., 1997). Since CtrA alone cannot account for the essential nature of ClpXP we postulate
one or several additional substrates (marked with '?') for the protease that have to be degraded to allow cells to proceed
through the cell cycle. The observed cell-cycle arrest of the clp mutants suggests that stabilization of the additional
substrate(s) result in a replication block similar to the one in C.crescentus cells expressing a stable and constitutive CtrA
protein. The additional ClpXP substrates could fall into two groups: (i) proteins that do not affect activity of CtrA and upon
stabilization lead to a CtrA-independent cell-cycle arrest; and (ii) proteins that affect CtrA activity [i.e. components of the
phosphorelay that results in phosphorylation of the CtrA protein (Wu et al., 1998)] and upon stabilization would lead to
increased concentrations of phosphorylated CtrA protein. Increased levels of phosphorylated CtrA combined with an increased
stability of CtrA would then account for the G1 cell-cycle arrest observed in clp mutants.
Post-Translational Control -- Conformation
Changes in the conformation of a regulatory protein
lead to changes in gene expression
Example: E. coli iron response--
Fur -- ferric uptake regulator
a transcriptional repressor that controls the
expression of numerous genes involved in
iron uptake and storage.
Here iron is acting as ____________?
Take home message:
Gene expression can be controlled post-translationally
by proteolysis and changes in conformation
of regulatory proteins
Regulation of gene expression
RNA polymerase
Regulatory proteins aa-tRNAs
RNA polymerase
DNA
Promoter Attenuator
operator
Transcription
Stop signal
Transcriptional control
(a) Transcription initiation:
positive/negative
(b) Transcription termination:
attenuation/anti-termination
Regulatory proteins
Antisense RNAs
mRNA
Ribosome
binding
site
Translation
Stop signal
Translational control
Translation initiation:
positive/negative
Degradation
Protein
Post-translational control
(e.g., proteolysis)
Other things we should think about in terms of regulation
Sigma factors: How do they work
and how are they regulated
Sigma factors: How do they work?
Most bacteria encode multiple sigma factors
(some of which are specialized and only used at certain times).
Each binds a different promoter consensus sequence
Alignment showing sigma 70 promoter conservation
The 4 major sigma factors of E. coli :
Sigma70
Primary sigma factor, or housekeeping sigma factor.
Encoded by rpoD .
When bound to RNAP Core allows recognition of -35 and -10 promoters.
No other factors required for RNAP binding and transcription initiation.
Sigma54
Alternative sigma involved in transcribing nitrogen-regulated genes (among others).
Encoded by rpoN (ntrA).
When bound to RNAP Core allows recognition of different -26 -12 promoters.
Requires an additional activator to allow open complex formation for transcription.
Sigma32
Heat shock factor involved in activation of genes after heat shock.
Encoded by rpoH (htpR).
Turned on by heat shock (either at the transcription or protein level).
Activates multiple genes involved in the heat shock response.
SigmaS (sigma38)
Stationary phase sigma factor.
Encoded by rpoS .
Turned on in stationary phase.
Activates genes involved in long term survival, eg. peroxidase.
Some sigma factors are always expressed but inactive without
a coactivator
Some are only expressed when the genes they regulate are required
Some are expressed but degraded
Some are expressed but inactive due to anti-sigma factors
Anti-sigma Factors

Anti-
Anti-sigma factors tightly (yet reversibly) bind sigma factors
and prevent their activity.
How might anti-sigma factors regulated?
Anti-sigma factors and flagellar gene expression in Salmonella
Flagellar genes
are expressed in
3 major classes.
Class 1 genes=
regulatory proteins
Class 2 genes=
basal body and
hook components
Class 3 genes=
chemotaxis genes
and flagellin
fliA encodes 28
flgM
So, how are Class 3 genes ever expressed since FlgM
is expressed as a class 2 gene and acts as an anti-sigma factor?
fliA encodes 28
So, how are Class 3 genes ever expressed since FlgM
is acting as an anti-sigma factor?
Figure 5 Model for the regulation of the stress response alternative sigma factor, σB, in Bacillus subtilus. (A) The σB structural gene, sigB, is
transcribed in an eight-gene operon from a general "housekeeping" σA-dependent promoter. It is also autoregulated and will transcribe the
rsbV—rsbW—sigB—rsbX four-gene operon. RsbW is an anti-σB factor and its activity is modulated by the other members of the operon in
response to either energy stress (measured as a drop in ATP levels) or to environmental stress. (B) Under stress-free conditions, RsbW binds σB to
prevent σB-dependent expression of stress-response genes. RsbW is also a kinase and under stress-free conditions will phosphorylate and
inactivate its antagonist, RsbV. Under poor growth conditions, energy stress occurs, ATP levels drop, and unphosphorylated RsbV accumulates
and inhibits RsbW. σB is free to transcribe stress-response genes.
In the absence of environmental stress, RsbT is free to interact with RsbU phosphorylase to prevent it from dephosphorylating RsbV-phosphate;
under these conditions, RsbW is free to inhibit σB-dependent expression of stress-response genes. RsbT is also a kinase that will phosphorylate
and inactivate its antagonist, RsbS. In the presence of environmental stress, RsbX, a RsbS-PO4 phosphatase, is activated to dephosphorylate
RsbS-PO4. In addition, stress also induces dephosphorylation of RsbR. Unphosphorylated RsbS is free to interact with RsbT and/or RsbR,
disrupting the RsbT-RsbU complex. The net effect of this interaction is to titrate RsbR, RsbS, and RsbT. This frees RsbU to dephosphorylate
RsbV-PO4. Unphosphorylated RsbV is free to interact with RsbW, disrupting the RsbW-σB complex. σB is free to transcribe stress-response
genes.
So anti-sigma factors are also regulated:
1). Secretion from the cell
2). Sequestration by an anti-anti-sigma factor
3). Interaction with effectors
Assignment: Think about how you might identify
regulators of an anti-sigma factor
Quorum sensing and population density
as a means of regulation
What is quorum sensing?
Quorum sensing--the ability of bacteria to
communicate and coordinate behavior via
signaling molecules.
Autoinducers--the signaling molecules produced
and used for quorum sensing.
Intra-species communication--communication
among the same species
Inter-species communication--communication
between different species
Bacterial Communication
controls gene expression
Quorum sensing allows bacteria to monitor population density.
Why is this important?
Vs
Vs
Gene expression
program A
Gene expression
program B
Gene expression
program C
Quorum sensing regulates virulence gene expression
in many bacterial pathogens
Vibrio cholerae
cholera
Streptococcus pneumoniae
otitis media and pneumonia
Pseudomonas aeruginosa
cystic fibrosis complications
Staphylococcus aureus
abscesses and endocarditis
Escherichia coli
diarrheal disease
How does Quorum sensing work?
Autoinducer
Bacterial Cell
The local concentration of signal is low due to diffusion
At high cell densities the local concentration of Inducer becomes high
Bacteria sense this and alter gene expression
Gram negative and Gram positive bacteria use different
signaling molecules (Acyl Homoserine lactones vs peptides)
Quorum sensing in Gram negative bacteria:
The Vibrio fischeri paradigm
This marine bacterium colonizes the light organ of the Hawaiian
squid, Eupryumna scolopes in a symbiotic relationship. The bacteria
acquire nutrients and the light they produce helps protect the squid
from predation. Additionally, factors produced by the bacteria are
required for normal tissue development in the squid.
Koropatnick TA, Engle JT, Apicella MA, Stabb EV, Goldman WE, McFall-Ngai MJ.
Microbial factor-mediated development in a host-bacterial mutualism.
Science. 2004 Nov 12;306(5699):1186-8.
How do these bacteria produce light?
The lux genes encode components of Luciferase.
Quorum sensing comes into play in terms of regulation
of expression of those genes by LuxR and LuxI.
LuxI is the autoinducer (AI) synthase, which produces AHL.
AHL is freely diffusible across the cell membrane.
LuxR is the cytoplasmic AI receptor/DNA binding
transcriptional activator. It is active only when bound to AI.
Thus, as the amount of AI increases (due to increased population density)
more AI enters the cell, affects LuxR and activates its regulatory ability.
This type of signaling is Intra-species communication--you are
monitoring your own kind. How is this possible since many
different bacteria produce AIs? (Formally known as AI-1).
Each species produces a unique AI-1 molecule.
Unlike the Gram negative AHL, the AI-1 peptides produced by
Gram-positive bacteria are not freely diffusible across the membrane.
Transporters are required to move the peptides.
Staphylococcus aureus Agr system. agrD encodes the AI-1 peptide.
AgrB transports and processes the peptide. AgrC and AgrA act as
a two-component-like system that senses AI accumulation and then
regulates downstream gene expression.
This type of signaling is Intra-species communication--you are
monitoring your own kind. How is this possible since many
different bacteria produce AIs?
Each species produces a unique AI-1 molecule.
What about Inter-species communication?
This is made possible by the production of AI-2 by the LuxS synthase.
Activity involves a phosphorelay system. At low cell density, LuxQ and LuxN
autophosphorylate, pass the phosphate to LuxU, which subsequently passes it to LuxO, which
then acts as a Response regulator and positively regulates expression of sRNA and Hfq.
These factors inhibit LuxR, which is required for LuxCDABE expression. At high cell
density (ie high concentrations of AI-1 and AI-2), LuxQ and LuxN act as phosphatases and
the system (phosphorelay) is reversed.
As if that wasn’t complicated enough, a third system has recently
been identified in multiple species of Vibrio!!!!
Questions?