Systems Microbiology
Wednesday Oct 11 - Ch 8 -Brock
Regulation of cell activity
• Transcriptional regulation mechanisms
• Translational regulation mechanisms
• Bacterial genetics
Image removed due to copyright restrictions.
See Figure 8-1 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Regulation of prokaryotic transcription
1. Single-celled organisms with short doubling times must respond extremely rapidly to their
environment.
2. Half-life of most mRNAs is short (on the order of a few minutes).
3. Coupled transcription and translation occur in a single cellular compartment.
Therefore, transcriptional initiation is usually the major control point.
Most prokaryotic genes are regulated in units called operons (Jacob and
Monod, 1960)
Operon: a coordinated unit of gene expression consisting of one or more related genes and
the operator and promoter sequences that regulate their transcription. The mRNAs thus
produced are “polycistronic’—multiple genes on a single transcript.
Regulatory Sequences
Genes Transcribed as a Unit
Promoter
DNA
Activator
Binding Site
A
B
C
Repressor
Binding Site
(Operator)
Figure by MIT OCW.
Initiation of transcription begins with promoter binding by
RNAP holoenzyme
holoenzyme = RNAP core + Sigma
Diagram of RNA polymerase and transcription removed due to copyright restrictions.
Brock Biology of Microorganisms, vol. 11, Chapter 7
Alternate Sigma Factors
recognize promoters of different architecture –
different regulons of genes
Table and graphs removed due to copyright restrictions. See Ishihama. Ann Rev Microbiol 54 (2000): 499-518.
Ishihama, 2000, Ann. Rev. Microbiol. 54:499-518
Transcription factors interact with different components of
RNAP in addition to sigma factor…..
Diagram removed due to copyright restrictions. See Ishihama. Ann Rev Microbiol 54 (2000): 499-518.
Ishihama, 2000, Ann. Rev. Microbiol. 54:499-518
Microbiology 151 (2005), 3147-3150; DOI 10.1099/mic.0.28339-0
Genome update: sigma factors in 240 bacterial genomes
Types of sigma factors…
Graphs removed due to copyright restrictions.
Transcription factors
Typically DNA binding proteins that associate with the regulated
promoter and either decrease or increase the efficiency of
transcription, repressors and activators, respectively - A
significant number of regulators do either one depending on
conditions
Domain containing protein-protein contacts,
holding protein dimer together
DNA binding domain fits in major grooves
and along phosphate backbone
5'
3'
TGTGTGGAATTGTGAGCGGATAACAATTTCACACA
ACACACCTTAACACTCGCCTA T TGTTAAAGTGTGT
3'
5'
Inverted repeats on the DNA
Figure by MIT OCW.
In presence of arginine, arginine biosynthesis genes are repressed
Repression
Relative Increase
Arginine Biosynthesis
No. cells
Total protein
Arginine added
Repression
Enzymes involved
in arginine synthesis
Induction
Time
Figure by MIT OCW.
In the presence of lactose, lactose metabolizing genes are induced
Induction
Relative Increase
Lactose Degradation
No. cells
Total protein
β-Galactosidase
Lactose added
Time
Figure by MIT OCW.
The metabolism of lactose in E. coli & the lactose operon
Galactoside permease
Lactose
•To use lactose as an energy source, cells must contain
the enzyme β-galactosidase.
•Utilization of lactose also requires the enzyme lactose
permease to transport lactose into the cell.
•Expression of these enzymes is rapidly induced
~1000-fold when cells are grown in lactose compared
to glucose.
Outside
Inside
CH2OH
HO
H
CH2OH
O
H
H
OH
H
H
OH
O
H
O
OH
H
OH
H
H
OH
Lactose
H
b-galactosidase
CH2OH
HO
H
Trans-glycosylation
O
H
OH
H
H
OH
CH2
O
H
H
HO
O
OH
H
OH
H
H
OH
Allolactose
H
LacZ: β-galactosidase; Y: galactoside permease;
A: transacetylase (function unknown).
P: promoter; O: operator.
LacI: repressor; PI and LacI are not part of the operon.
b-galactosidase
CH2OH
HO
H
O
H
OH
H
H
OH
Galactose
CH2OH
OH
H
+
H
HO
O
H
OH
H
H
OH
IPTG: nonmetabolizable
artificial inducer
(can’t be cleaved)
OH
H
Glucose
figure by MIT OCW.
Negative regulation of the lac operon
lac operon
RNA polymerase
Negative regulation: The product
of the I gene, the repressor,
blocks the expression of the
Z, Y, and A genes by
interacting with the operator
(O).
The inducer (lactose or IPTG) can
bind to the repressor, which
induces a conformational
change in the repressor,
thereby preventing its
interaction with the operator
(O). When this happens,
RNA polymerase is free to
bind to the promoter (P) and
initiates transcription of the
lac genes.
DNA
I
P
O
Z
Y
A
mRNA
lac repressor
Lactose
Medium
Thiogalactoside
transacetylase
Lactose
permease
β-galactosidase
How do we know that lacI encodes a trans-acting repressor?
Nascent
polypeptide
chains
Figure by MIT OCW.
Symmetry matching between the tetramers of lac repressor and the
nearly palindromic sequence of the lac operator
Each monomeric unit of lacI is 37-kD
-10
+1
+10
+20
+30
5' ATGT TGTGTGGAATTGTGAGC GGATAACAATTTCACACAGGAA 3'
3' TACAACACACCTTAACACTCGC CTATTGTTAAAGTGTGT CCTT 5'
Half-site
Figure by MIT OCW.
Image of the X-ray structure of lac repressor tetramer bound to two
21-bp segments of DNA removed due to copyright restrictions.
The lac operator sequence is a nearly perfect
inverted repeat centered around the GC base
pair at position + 11.
“Diauxic” growth preferential use of one carbon source over another, in sequential fashion
Number of Viable Cells
Lactose exhausted
Glucose exhausted
Glucose
and lactose
added
Growth on
lactose
Growth on glucose
Time of Incubation (hr)
Figure by MIT OCW.
Regulation of the lac operon involves more than a simple on/off
switch provided by lacI/lacO
Observation: Glucose is a preferred sugar for E. coli, which uses glucose
and ignores lactose in media containing both sugars. In these cells, β
galactosidase level is low, suggesting that derepression at the operator site
is not enough to turn on the lac operon. This phenomenon is called
catabolite repression.
This involves regulation of cyclic AMP (cAMP) levels, and its interaction
with the Catabolite Activator Protein, CAP.
cAMP-CAP complex activates lac gene expression.
Cooperative binding of cAMP-CAP and RNAP on the lac promoter
RNA polymerase
a submit
cAMP-CAP
-84
-50
CAP site
+1
Polymerase site
Lac control
region
Figure by MIT OCW.
cAMP-CAP contacts the α-subunits of RNAP and enhances the binding of RNAP
to the promoter.
X-ray structure of CAP
cAMP bound to DNA
Image of the X-ray structure of the CAP-cAMP dimer in complexwith DNA removed due to copyright restrictions.
Catabolite control of the lac operon
(A)
High glucose
Inactive adenylate cyclase
No cAMP
ATP
(B)
(a) Under conditions of high glucose, a
glucose breakdown product inhibits the
enzyme adenylate cyclase, preventing the
conversion of ATP into cAMP.
(b) As E. coli becomes starved for glucose,
there is no breakdown product, and
therefore adenylate cyclase is active and
cAMP is formed.
Active adenylate
cyclase
Low glucose
ATP
cAMP
(C)
cAMP
cAMP
+
CAP
CAP
(c) When cAMP (a hunger signal) is
present, it acts as an allosteric effector,
complexing with the CAP dimer.
cAMP
(D)
CAP
P
O
Z
Y
A
Figure by MIT OCW.
CAP sites are also present in other promoters. cAMPCAP is a global catabolite gene activator.
(d) The cAMP-CAP complex (not CAP
alone) acts as an activator of lac operon
transcription by binding to a region within
the lac promoter. (CAP = catabolite
activator protein; cAMP = cyclic adenosine
monophosphate)
Positive and negative regulation of the lac operon
Glucose present (cAMP low); no lactose
CAP
Z
Promoter
Y
A
Operator
A
Repressor
Glucose present (cAMP low); lactose present
CAP
Z
Y
A
Very little lac mRNA
Inducer-repressor
Lactose
B
No glucose present (cAMP high); lactose present
cAMP
Z
C
Y
A
Abundant lac mRNA
Figure by MIT OCW.
Transcriptional termination can be an important target
for regulation
5'
3'
3'
5'
Termination site reached;
chain growth stops
5'
3'
5'
3'
3'
Release of polymerase
and RNA
5'
5'
Figure by MIT OCW.
The tryptophan trp operon: two kinds of negative regulation
Structural genes
Control sites
trp,O
mRNA
(low trp levels)
Leader
mRNA
(high trp levels)
Attenuator
trpL
trpE
trpD
trpB
trpC
trpA
or
Promoter
Operator
Tryptophan + trp repressor dimer
Trp-repressor complex activated
for DNA binding
+1
-35
Inactive repressor
Start of transcription
Tryptophan
RNA polymerase
Active repressor
Binds Operator; blocks RNAP binding &
represses transcription;
Tryptophan a co-repressor
mRNA
Genes are ON
Genes are OFF
Figure by MIT OCW.
Image of trp and DNA removed due to copyright restrictions.
Many genes terminate transcription at sequences
downstream of the coding sequence
Image removed due to copyright restrictions.
See Figure 7-32 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Attenuation and the
Tryptophan Operon
Image removed due to copyright restrictions.
See Figure 8-24 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Attenuation is mediated by the tight coupling of transcription
and translation
•The ribosome translating the trp leader mRNA follows closely behind the RNA polymerase
that is transcribing the DNA template.
•Alternative conformation adopted by the leader mRNA.
High tryptophan
Transcription
terminator
Leader
peptide
completed
1
trpL mRNA
4
3
2
+
UUU 3
,
"Terminated"
RNA polymerase
Ribosome transcribing
the leader peptide mRNA
and blocking sequence 2
Low tryptophan
Incomplete
leader peptide
Antiterminator
2
1
Ribosome stalled
at tandem Trp codons
trp operon mRNA
3
Transcribing RNA polymerase
4
•The stalled
ribosome is waiting
for tryptophanyltRNA.
•The 2:3 pair is not
an attenuator and is
more stable than
the 3:4 pair.
DNA encoding
trp operon
Figure by MIT OCW.
•
•
The arabinose (ara) operon
3 genes encoding enzymes of the arabinose degradation pathway
arabinose
–
–
–
(1) araA => L-arabinose isomerase
(2) araB => L-ribulose kinase
(3) araD => L-ribulose 5-phosphate
arabinose
1
Regulatory elements
–
–
–
araO1, araO2
araI (I for inducer)
PBAD promoter
L-ribulose
2
L-ribulose –5-P
3
D-xylulose –5-P
Pentose P Pathway
The arabinose (ara) operon of Escherichia coli
t
P
P
t
CRP
O2 O1
araC
I1 I2
araB
araA
araD
AraC dimer
Activator/
Repressor
Interaction of AraC with O and I sites determines transcription at
the Ara locus
Key features of Arabinose regulation
1. Arabinose is a positive regulator of transcription.
2. In the absence of arabinose AraC binds regulatory sites I1 and O2.
This represses AraBAD operon transcription (DNA looping).
3. AraC levels are autoregulated by excess AraC binding to O1
4. When arabinose is bound to AraC, AraC binds I1 and I2.
5. When Glucose is absent, cAMP levels rise and cAMP-CRP bind
adjacent to I1.
Together these events trigger AraBAD expression.
ara Transcriptional control-1
t
P
O1 CRP
O2
I1 I2
araC
P
araB
t
araA
araD
1. No L-arabinose
O2
O2
AraC dimer is flexible
P araBAD
P araC
Active
repressed
O1
I1 I2
AraC binds to O2 and I1 repressing P araBAD
P araC
P araBAD
repressed
repressed
O1
I1 I2
Excess AraC binds to O1 regulating cellular
levels of AraC protein
ara Transcriptional control-2
t
O2
P
O1 CRP
P
t
I1 I2 araB araC
araA
araD 2. + Inducer L-arabinose
+ cAMP-CRP
t
O2
P araBAD induced
(activated)
P
O1
I1 I2
AraC binds to I2 and I1 activating P araBAD
Differences between the arabinose and lactose operons
1. AraC can act as both a repressor and activator of AraBAD expression.
This is an example of positive control.
2. AraC can regulate its own synthesis by repressing its own transcription
This is a common feature of many genes.
3. Ara operon provides an example of regulation at a distance by DNA
looping.
Common mechanisms of regulation of transcription,
with variations on a theme…
lac
ara
trp
Regulation
negative
positive
negative,
positive, auto-
negative attenuation
Operators
CRP-site
Regulator
(location)
lacO1, O2, O3
araO1, O2, I
trpO
+
+
-
lacI
upstream
araC
upstream
allolactose
(activator)
arabinose
(activator)
Effector
trp R
distant
tryptophan
(co-repressor)
“Flipping promoters” Flagellar phase variation
Flagellar
Phase variation
Figure by MIT OCW.
Environmentally-responsive adaptation
External stimuli
Transmembrane
receptor
Indirect
effect
Internal stimuli
Physiological
change
Membranepermeable
signals
Gene
Regulation
Simple paradigm for environmental signalling – the twocomponent system
> 30 such systems in E. coli – also found in plants and fungi
Image removed due to copyright restrictions.
Basic model for a two component-regulatory system
Sensor histidine kinase (HK) – may or may not be transmembrane –
phosphorylates itself
Response regulator (RR) – often, but not always affects gene expression –
phosphorylated by HK
Hoch and Varughese, 2001, J. Bacteriol. 183:4941-4949
The PhoR/PhoB two-component regulatory system in E. coli
Diagram removed due to copyright restrictions.
In response to low phosphate
concentrations in the environment
and periplasmic space, a phosphate
ion dissociates from the periplasmic
domain of the sensor protein PhoR.
This causes a conformational change
that activates a protein kinase
transmitter domain in the cytosolic
region of PhoR. The activated
transmitter domain transfers an ATP
γ-phosphate to a histidine in the
transmitter domain. This phosphate
is then transferred to an aspartic acid
in the response regulator PhoB.
Phosphorylated PhoB then activate
transcription from genes encoding
proteins that help the cell to respond
to low phosphate, including phoA,
phoS, phoE, and ugpB.
Two-components alone aren’t always sufficient –
phospho relays are through multiple protein modulators
are a common regulatory mechanism
Diagram removed due to copyright restrictions.
Stock et al. 2000, Ann. Rev. Genet 69:183-215
‘phosphorylation cascades’
Allow response
to wide range of
chemical and
physical stimuli
Diagram removed due to copyright restrictions.
Many variations on
the basic theme
exist and the more
they are studied
the more
permutations are
observed
Stock et al. 2000, Ann. Rev. Genet 69:183-215
Attractants
Transducer (MCP)
CheW
CheA
ATP
CheW
CheA
P
Flagellar
motor
+CH2
CheP
CheB
P
CheB
CheY
CheY
P
CheZ
-CH2
Cytoplasmic
membrane
Cell wall
Figure by MIT OCW.
Quorum Sensing
Image removed due to copyright restrictions.
See Figure 8-23 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
NADPH + H+
hν
FMN
NAD(P)H + H+
Oxidoreductase
NAD(P)+
ATP
RCOOH
Luciferase
FMNH2
O2
Fatty Acid Reductase
RCHO
AMP+
PPi
NADP+
RCHO + FMNH2 + O2
Luciferase
RCOOH + FMN + H2O + hν
Substrates, Products and Pathways Involved in the Bacterial Bioluminescence Reaction
Figure by MIT OCW.
1013
1011
MJ-1
Luminescence
(
Quanta/sec
OD660
(
1012
1010
pJE202
pJE201
pJE205
109
108
Growth
0
.2
.4
.6
OD660
.8
1.0
Figure by MIT OCW.
Nucleic Acids Res. 1987 December 23; 15(24): 10455–10467.
Nucleotide sequence of the regulatory locus controlling expression
of bacterial genes for bioluminescence
Negative
Feedback
lux R
Operon L
Positive
Feedback
lux I
Model for Regulation of
Bioluminescence
lux CD ABE
Operon R
Regulatory Proteins
Enzymes for
Bioluminescence
Figure by MIT OCW.
Vibrio fischeri and luminescence :
LuxR
LuxI
LuxR
Target Genes
Figure by MIT OCW.
Quorum Sensing
O
H
R
C
H
O
CH2
C
N
O
H
Acyl Homoserine Lactone (AHL)
Figure by MIT OCW.
Image removed due to copyright restrictions.
See Figure 8-22b in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
Examples of bacteria that use acylated homoserine lactones
Bacteria
Function
Vibrio fischeri
luminescence
Aeromonas hydrophila
proteases
Agrobacterium tumefaciens
conjugation
Burkholderia cepacia
siderophores
Chromobacterium violaceum
antibiotics
Erwinia chrysanthemi
pectinase
Pseudomonas aereofaciens
phenazines
Pseudomonas aeruginosa
biofilms, etc
Rhizobium etli
number of nodules
Yersinia pseudotuberculosis
agreggation and motility
Variations and Complications :
Vibrio harveyi
LuxP
LuxN
H
H
P
D
P
LuxQ
D
LuxU
P
H
LuxLM
P
P
D
X
LuxS
LuxO
LuxR
luxCDABE
Figure by MIT OCW.
Image removed due to copyright restrictions.
See Figure 8-1 in Madigan, Michael, and John Martinko. Brock Biology of Microorganisms.
11th ed. Upper Saddle River, NJ: Pearson Prentice Hall, 2006. ISBN: 0131443291.
The “stringent response”
A type of translational global control
When bacteria are starved of nutrients, they immediately
shut down gene expression and other metabolic activities.
1. Total RNA synthesis is reduced to ~ 10% of normal levels.
2. There is a massive >10-fold reduction in rRNA and tRNA
transcription.
3. Protein synthesis decreases.
(The unusual nucleotides ppGpp and pppGpp accumulate
during the stringent response).
Stringent response in E. coli
1. Binding of an uncharged
tRNA to the A-site
2. Binding of RelA to the 30S
subunit
3. Synthesis of ppGpp
4. Downregulation / inhibition of
transcription
Image removed due to copyright restrictions.
The “stringent response”
A type of translational global control
OH
HO
P
O
O
OH
O
P
O
O
O
NH
N
P
O
OH
CH2
N
N
NH 2
O
OH O
HO
P
O
O
O
P
OH
OH
The ppGpp inhibits the elongation phase of transcription.
The stringent factor RelA is a pppGpp synthetase that is
associated with ~ 5% of ribosomes.
RelA protein produces one pppGpp every time the A site is
occupied by an uncharged tRNA.
The “stringent response”
A type of translational global control
Ribosomal protein L11 undergoes a conformational change
when an uncharged tRNA binds.
This activates RelA stringent factor.
The unusual nucleotides ppGpp and pppGpp accumulate
during the stringent response.
Total RNA synthesis is reduced to ~ 10% of normal levels.
There is a massive >10-fold reduction in rRNA and tRNA transcription.
Protein synthesis decreases.
(The SpoT protein degrades ppGpp so that normal gene
expression can resume rapidly when conditions improve.)
Review of gene regulation in bacteria.
With genes that are expressed constitutively, promoter strength determines the level of expression.
DNA-binding proteins can switch genes on and off.
Repressors switch genes off.
[They prevent RNA pol from gaining access to promoters].
Activators switch genes on.
[They enable RNA pol to bind to promoters]
Activity of repressors and activators can be influenced by small molecules, temperature, phosphorylation etc.
RNA secondary structure can control gene expression.
With attenuation, AA-tRNA availability influences
early termination.
With riboswitches, small molecules influence
early termination or translation intiation.
Alternative sigma factors bring about global changes
in gene expression.
The stringent response shuts down gene expression
when times are tough.
Promoter inversion can affect gene expression in
pathogenic bacteria.