Review of simple regulation

1
Combinatorial control of a bacterial metabolic
promoter
Review of simple regulation Introduction to
combinatorial control A prokaryotic example The
complex promoter acsP2 and its activation by
CRP Introduction to nucleoid proteins (e.g. Fis
and IHF) Fis anti-activates CRP-dependent
activation How does IHF inhibit CRP-dependent
activation? The experimental approach The
mechanism (results and working model) Unanswered
questions
2

• Bacterial cells use diverse mechanisms to
  regulate transcription
• Sigma factors partition limited RNA polymerase
  (RNAP)
• to different promoter subsets

3
Bacterial cells use diverse mechanisms to
regulate transcription 2. Transcription factors
alter binding of RNAP to promoter
4
Weak promoters Regulated by activation
Weak promoter 1. fewer elements 2. less consensus
-35
UP
-10
5
Recruitment by TF that increases affinity
?
? ?
?NTD
?
?CTD
-35
UP
-10
TF
Interactions that enhance transcription initiation
6
Repression by too much affinity
?
? ?
?NTD
?
?CTD
-35
UP
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TF
Interactions that enhance transcription initiation
RNAP cant break contacts used for TCC TOC
formation
7
Combinatorial Control A simple example
TF1
TF1
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-10
8
Combinatorial Control a textbook,
eukaryotic example
9
Combinatorial Control a prokaryotic example
10
Acetate Switch
Glucose
2 NAD
2 NADH 2 H
2 ADP
2 ATP
Pyruvate
CoASH NAD
CO2 NADH H
Acetyl-CoA
AMP
ACS
CoASH
AcetylP
Acetyl-AMP
PPi
ACS
ATP
Acetate
ACS acetyl-CoA synthetase
11
Regulation of Acs activity
Acetyl-CoA
AMP
Acs
CoASH
AcAMP
PPi
Acs
ATP
Acetate
12
Regulation of acs transcription
TCA
acCoA
Acs
GROWTH
ace
TIME
13
P1
P2
pnrfA
acs
actP
nrfA
nrfB
nrfC
yjcH
-180
-226
-267
-153
-98
-59
IHF II
IHF I
FIS I
IHF III
FIS II
FIS III
P2
1
CRP I
CRP II
P1
-69.5
-122.5
Pnrf
14
Beatty et al., 2003
acs transcription CRP (cAMP Receptor
Protein)-dependent proximal P2 is the major
promoter
acs
S70
? ??

1
CRP I -69.5
CRP II -122.5
P2
Higher affinity CRP I absolutely required for any
activation Lower affinity CRP II doubles activity
with a twist!
15
Bacterial cells use diverse mechanisms to
regulate transcription 3. Regulation by
histone-like proteins that organize bacterial
chromatin
16
Bacterial chromosome The traditional view
A tangled knot of DNA
Dr. Gopal Murti Science Photo Library Photo
Researchers
17
Bacterial chromosome The modern view Kim et al.,
2004
18
12 histone-like (nucleoid) proteins in E.
coli Relative amounts change with phase of growth
Fis (Factor for Inversion Stimulation) predominat
es during EP undetectable by ESP IHF
(Integration Host Factor) present during
EP Increases 4X 2nd most predominate in ESP
19
Nucleoid proteins influence acs transcription
P2

• Fis
• IHF
• Browning et al., 2004

20
Factor for Inversion Stimulation (Fis)
homodimer bends DNA 50-90o
Hengen et. al., 1997
21
Fis sites overlap CRP sites Do they compete?
22
Fis and CRP interactions DNase I footprint
analysis
Fis out-competes CRP Fis CRP resembles Fis alone
23
Regulation of acs transcription
GROWTH
TIME
24
Anti-activation Fis sterically hinders CRP
25
Integration Host Factor (IHF)
W W W W W W n n n n n n n n W A T C A A n n n
n T T R T A T T T T a c a t g c a c
T t a C A A t t g a T T A I A
g T A T c t t c c t c t t T t T C A
A c a g c a T G II c A T A A c
t g c a t g t t c c T C A A a g a a T T A
III
26
IHF binds simultaneously with FIS and CRP DNase I
footprint analysis
27
Both FIS and IHF inhibit CRP-dependent
activation 2 distinct mechanisms
28
Approach
29
How do you monitor promoter activity?reporter
assay
lac Z
P2
CRP I
30
How do you monitor promoter activity?reporter
assay
Activity (Miller Units)
OD590
31
Acs Regulatory Region Nested Deletions
FIS III
FIS II
CRP I
FIS I
CRP II
IHF II
IHF I
IHF III
WT / 2
WT
WT / 6
WT
32
Acs Regulatory Region Mutations
CRP I
CRP II
IHF III
WT / 2
WT Activity
WT/6
(CRP and IHF dependent)
WT Activity
33
Acs Regulatory Region Summary
Activating
Enhancing
IHF III
CRP I
CRP II
Activation requires CRP I, enhanced by CRP
II Inhibition of activation requires IHF III
CRP II
34
Mechanism
35
Proteins have surfaces Rhodius
RNAP
AR 1
AR 1
Class I
36
Proteins have surfaces Rhodius
RNAP
Class II
37
Acs Regulatory Region Summary
Activating
Enhancing
IHF III
CRP I
CRP II
We can use our knowledge concerning the surfaces
of CRP and their interactions to determine
mechanism
38
The Experimental DesignComplementation
CRP
lac Z
P2
CRP I
39
Monitoring promoter activity of
pacs155reporter assay
Activity (Miller Units)
OD590
40
Monitoring promoter activity of pacs155a
simplified version
Activity (Miller Units)
41
pacs155 is AR1-dependent classic Class I behavior
RNAP
AR 1
AR 1
42
How can AR2 AR3 inhibit?
AR 2
RNAP
AR 1
AR 1
AR 3
Later
43
Key
pacs205
CRP I
CRP II
44
Key
pacs236
CRP I
CRP II
IHF III
45
Key
pacs236c2m
CRP I
CRP II
IHF III
Together AR2 and AR3 inhibit severely Like pacs236
46
Mechanism a working model
CRP I
CRP II
IHF III
CRP I is high affinity, CRP II is low affinity
Low concentration of CRP Fill ONLY CRP I
AR1-dependent, AR2-enhanced activation
47
Mechanism a working model
CRP I is high affinity, CRP II is low affinity
CRP I
CRP II
IHF III
High concentration of CRP Fill BOTH CRP I CRP
II AR2-dependent inhibition of AR1-dependent
activation
48
Regulation of acs transcription Depends on Fis
and CRP
IHF
GROWTH
TIME
49
The role of IHF To make the sense of regulation
( or -) dependent on CRP Depends on AR2?!
50
How can AR2 enhance or inhibit?
AR 2
RNAP
AR 1
AR 1
pacs205/236 have two dimers Four AR2 However,
pacs155 exhibits fundamental behavior Thus,
likely due to one of two AR2 on proximal dimer
51
How does IHF enslave CRP II?
CRP
CRP
IHF
P2
CRP I
CRP II
IHF III
AR2-dependent inhibition of AR1-dependent
activation In vitro evidence suggests that AR2
does not permit promoter clearance
52
Repression by too much affinity
?
? ?
?NTD
?
?CTD
-35
UP
-10
TF
Interactions that enhance transcription initiation
RNAP cant break contacts used for TCC TOC
formation
53
But, wait - - theres more!
P1
P2
pnrfA
acs
actP
nrfA
nrfB
nrfC
yjcH
-180
-226
-267
-153
-98
-59
IHF II
IHF I
FIS I
IHF III
FIS II
FIS III
P2
1
CRP I
CRP II
P1
-69.5
-122.5
Pnrf