LRET to Study ProteinProtein Interactions Sigma70 Binding to Core RNA Polymerase: Biochemistry and D

1
LRET to Study Protein-Protein Interactions-Sigma7
0 Binding to Core RNA Polymerase Biochemistry
and Drug Discovery
Richard R. Burgess, Bryan Glaser Veit Bergendahl,
Rob Chumanov University of Wisconsin-Madison McArd
le Laboratory for Cancer Research burgess_at_oncology
.wisc.edu Colorado State University June 12, 2009
2
Lecture Outline Bacterial RNA polymerase
and sigma factors Ordered fragment ladder
far-Western analysis Sigma binding site on core
RNA polymerase Protein-protein interaction as a
site for drug discovery Development of
fluorescence-based binding assay High-throughpu
t (HTS) screening Determining biochemical
binding constants
3
E. coli RNA Polymerase (the enzyme my lab studies)
Core Sigma Holoenzyme a2bbw
s a2bbws Subunit Gene MW Functio
n a rpoA 36 kDa specificity b rpoB 150
kDa catalytic b rpoC 155 kDa s
binding w rpoZ 10 kDa assembly s70 rpoD 70
kDa specificity
4
RNA Polymerase and Gene Expression
Promoter
DNA
- 35
- 10
holo
Promoter binding by RNA polymerase
?
DNA
?
?
?
?
Initiation and elongation of transcription
?
core
?
s released
DNA
?
?
?
Protein
Translation
Ribosome
mRNA
5
The Seven Sigma Factors of E. coli
K12 Factor Gene Size (kDa) Consensus
Binding Site Genes Regulated Discovered s70
(sD) rpoD 70 TTGACA-N17-TATAAT
Housekeeping 1969 s54 (sN) rpoN (ntrA)
54 CTGGCAC-N5-TTGCA Nitrogen
metabolism 1985 sS rpoS (katF)
38 TTGACA-N12-TGTGCTATACT Stationary phase
1989 s32 (sH) rpoH (htpR) 32
CTTGAA-N14-CCCCATNT Heat shock
1984 sF (s28) fliA 28
TAAA-N15-GCCGATAA Flagellar proteins
1987 sE rpoE 24 GAACTT-N16-TCTGA
Extreme heat shock 1989 sfecI fecI
19 GGAAAT-N17-TC Iron
transport 1995
6
Competition for Core Binding
Housekeeping
7
Which region of core does s70 interact with?
b
b
a
a
?
8

Preparing an Ordered Fragment Ladder
Cloning
Overexpression of His-tagged protein
Isolate, wash, and solublize inclusion body
Chemical Cleavage
interaction
cleavage
partner
site
X
X
X
1
2
3
4
His
2
- chelate column
Purification on Ni
a
4
3
2
1
His
3
2
1
1
2
1
SDS-PAGE / Western blot
3
1
2
4
a
b
c
d
9
(No Transcript)
10


C Terminus
N Terminus
NT63
152
N-His
?
146
123
104
92
50
30
17
?
C-His
16
137/136
123
103
61
48
29
6
11

Mapping ? Epitopes by Ordered Fragment Ladder
Western

115
236
409
495
686
868
1020
1295
NT15
7RC11
7RC74
7RC78
7RC80
NT73
900
1100
700
1300
100
300
500
'
A
E
F
G
?
B
C
D
H
Zn-finger
RNA
motif
3' end
12
(No Transcript)
13
Radio-labeling HMK Probe
14
Localization of ?70 Binding Sites on ? and ? by
Far-Western Blotting
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8 9 10 11
Coomassie stain
far-Western
? 70
700
900
1100
1300
100
300
500
?
A
B
C
D
E
F
G
H
Zn-finger
RNA
motif
3' end
15
b coiled-coil
Structure of Thermus aquaticus core RNA polymerase
(Zhang, Campbell, Minakhin, Richter, Severinov,
Darst, Cell 98, 811-824, 1999)
16
260 270
280 290
300
E. coli T. aq.
535 545
555 565
575
N266
N309
R275
E295
A302
R297
R293
Y269
Q300
K280
17
Region 2.2
X-ray Crystal Structure of E. coli Sigma70, amino
acids114-448 (Malhotra, Severinova, and Darst
Cell 87, 127-136, 1996)
18
In silico modeling Protein-protein docking
experiments

• Hex docking software
• Uses geometric, hydrophobic, and electrostatic
  fitting to model the interaction between two
  unbound proteins.
• Spherical polar Fourier correlations (overlapping
  surface skins)

(Larry Anthony, 2001)
19
Mutations that disrupt ?70-core binding In
beta prime 260-309 (Arthur Burgess) In ?70
regions 2.1 and 2.2 (Sharpe Gross) R275Q
V387I R293Q D403N E295K
E407K R297S G408D A302D
20
b260-330 s70 region 2.1-2.2 docking model
(Burgess and Anthony, Cur Opinions Microbiol,
2001)
21
Structure of Holo RNA Polymerase
-10
Thermus thermophilus (Vassylyev DG et al.
Nature, 417 712-9, 2002)
22
The bcoiled-coil Peptide is an Attractive Target
for Drug Discovery
1. This region represents the primary binding
site on core RNA polymerase for all sigma
transcription factors. Thus it is a prime target
for interference with basic cell metabolism. 2.
It would be difficult for the cell to become
resistant to a drug mimicking this peptide. It
would have to accumulate mutations in genes for
all 7 sigmas. 3. This region is highly conserved
among all of the over 65 bacteria sequenced so a
drug binding to it could represent a very broad
spectrum antibiotic. This peptide binds the major
B. subtilis sigma. 4. This sequence is not found
in human nuclear or mitochondrial RNA polymerases
so a drug that interacts with it would not be
expected to affect human transcription.
23
Alignment of various prokaryotic and eukaryotic
b subunit fragments
265 284
292 309
Helix
Helix
K P
D P
S L
directed mutations
D
L
D
D
A
Q
E
Q
K
E
D
D
E.c. B.s. B.a. H.i. S.a. B.b. M.t. T.a. H.s.
Escherichia coli Bacillus subtilis Bacillus
anthracis Haemophilus influenzae Staphylococcus
aureus Borrelia burgdorferi Mycobacterium
tuberculosis Thermus aquaticus Human RpbI









24
Luminescence Resonance Energy Transfer
LRET
Veit Bergendahl Bryan Glaser Tomasz Heyduk Larry
Anthony Rob Chumanov Richard Burgess
As a Tool to Monitor Protein-Protein Interactions
High Throughput Screening for a new Class of
Antibiotics
25
FRET - Fluorescence Resonance Energy
Transfer LRET - Luminescence Resonance Energy
Transfer
lexcite
l1
F2
(Acceptor)
F1
(Donor)
Beta Coiled-coil Fragment
Drug candidate
Free Sigma70
l2
lexcite
l1
F1
F2
Beta Coiled-coil Fragment
l1
Bound Sigma70
26
The Dyes
AMCA-DTPA-Maleimide Eu3
cs124-DTPA-Phe-NCS Tb3
s 70 (C132S, C291S, C295S S442C)
7-amino-(diethylene-triamine-pentateic acid)
-4-methyl-coumarin-3-methylamide-ethylenediamine-N
-2-ethyl-maleimide (Eu3-chelate)
Carbocystryl24-diethyletriamine-pentaacetate-pheny
lalanine-p-isothiocyanate (Tb3-chelate)
HMK-His6- b-100-309 (C198)
IC5-PE- Maleimide
IC3-Oxo- Succinimide
N-Ethyl-N'-5-(N"succinimidyloxycarbonyl)pentyl i
ndocarbocyanine chloride
N-Ethyl-N'-5-N''-(2-maleimidoethyl)piperazinocar
bonylpentyl -3,3,3',3'-tetramethyl-2,2'-indodica
rbocyanine
27
Derivatization of Proteins
A. Reaction of a thiols with a maleimide
28
Overlap of Tb-emission and IC3-Absorption
29
Purification and Derivatization of theRNAP
b-Fragment
Bergendahl, V, Anthony, LC, Heyduk, T,
and Burgess, RR, 2002 Anal Biochem. 307 368-74
30
EMS-Competition-Assays with b(100-309)

b-fragment concentration
Coomassie-stain
b-s70- complex
s 70
UV-screen (Eu-emission by labeled s70)
b-s70- complex
s 70
Storm system (IC5-emission by labeled bfragment)
b-s70- complex
31
LRET-Assays
with the
Multiplatereader
32
LRET Assay Competition
s
s
33
Screening of ChemBridge Library - 16,000 Compounds
250 potential Hits (gt65 inhibition at 3 mM) 30
were selected after exclusion of false positives
(identified by quenching of the donor signal) 4
hits were confirmed by in vitro transcription
(IC50 lt 40 mM)
A) raw data (384 well, 30 µl)
B) initial hit validation
(in vitro transcription)
34
Four lead compounds being characterized further
35
The LRET Assay Properties and Uses
Bergendahl, V, Heyduk, T, Burgess, RR 2003 Appl
Environ Microbiol. 69 1492-98
- Robust assay with respect to substrate
stability and solvents - Assay conditions30-200
µL total volume, 100 mM NaCl, 50 mM, Tris-HCl, 5
glycerol, 20 nM s70, 20 nM b'-fragment and 2.5
DMSO (when library samples are used) - Highly
sensitive (limit of detection 200 fmol label) -
Large signal-to-noise ratio (gt10 at maximum
signal) - 96 or preferably 384-well format

• It is ideally suited for high through-put
  screening to find lead chemical compounds for
  antibiotic development
• It is a superior homogeneous assay for binding
  studies
• The physical chemical parameters of sigma-core
  RNA polymerase binding

Veit Bergendahl, Larry Anthony, Bryan
Glaser, Brian Olson, Rob Chumanov
36
Random Labeling of Lysine Residues distances
between labeled residues in core RNAP and s70
Forster radius (65 A)
o
(Larry Anthony)
37
Protocol for Preparing Labeled Proteins for LRET

• Overproduce proteins in E. coli and purify
  protein (Protein is eluted in carbonate buffer
  (160 mM Na2CO3, 330 mM NaHCO3, pH 9.5)
• Concentrate protein 1 mg/ml
• Label lysines with 5-fold molar excess of dye to
  protein molecules
• Use size exclusion column to separate free dye
  from protein
• Dialyze into storage buffer (150 mM Kglu, 50 mM
  Tris HCl pH 7.9, 0.1 mM EDTA, 0.1 mM DTT, 50
  glycerol)
• Characterize labeled protein (usually get about
  1 dye/protein)

(Bryan Glaser)
38
Purified Proteins used in Studies

Tb-core cannot be visualized with fluorescent
scan
39
Sigma factors are near 100 active in binding
core RNA polymerase s70 and s32 were labeled
with IC3-OSu 50 nM s was incubated with 250 nM
core RNAP for 30 minutes at room temp Reactions
were electrophoresed on 12 native Tris-glycine
gels for 3 hours at 4oC Bands were visualized by
fluorescence on the Typhoon imager (Cy3 mode)
s70 s32 - -
core
(Larry Anthony, 2003)
40
Approach to Equilibrium Binding
100 mM NaCl, 220C
(Bryan Glaser)
41
Typical LRET Experiment

• Performed in 384-well plate
• Volume 30 mL
• Buffer 50 mM Tris-HCl, pH 7.9, 5 glycerol, and
  salt
• Donor Tb-labeled protein (Ex. 340 nm, Em. 490
  nm)
• Concentration held constant (2-20 nM)
• Acceptor Fluorescein-labeled protein (Ex. 490
  nm, Em. 520 nm)
• Concentrations up to 500 nM
• Background can increase above 200 nM
• For equilibrium studies Incubated at 22?C for 1
  hour.

42
Characterization of LRET Assay
43
Effect of Salt on s-Core Interaction
s70
s32
Conclusions ?70 more sensitive to NaCl
than to Kglu ?32 not very sensitive to
either salt
44
Summary of Characterization

• Labeled proteins are stored at 1 mg/mL and
  contain 1-2 molecules of dye per protein.
• All of the labeled sigma can bind core.
• Tb-core is slightly defective in binding of
  sigma.
• The LRET assay is very robust.
• The assay can tolerate high concentrations of
  solvents, detergents, and glycerol.
• The assay is sensitive to salt type and
  concentration, MgCl2, and BSA.

45
Modeling Interaction of s with Core RNAP
46
Interaction of s70 with Core RNAP
10 nM Tb-core (fit as 50 active)
47
Interaction of s32 with core RNAP
10 nM Tb-core (fit as 50 active)
48
Summary of Binding Studies
49
Interaction between Sigmas and Core Binding
Constants - Preliminary Results
KD?/KD?70 at 100 mM NaCl s32 0.1
s70 1.0 s54
1.2 sF 1.9 sE 1.5
sFecI 4.6 sS 6.9 Values are
averages of 2-3 determinations and are
/- about 20-30
(Veit Bergendahl)
50
s70 Interaction with bcc (a.a. 100-309)
10 nM Tb-bcc (100-309) (fit as 50 active)
51
s32 Interaction with bcc (a.a.100-309)
10 nM Tb-bcc (100-309) (fit as 50 active)
52
Comparison of KD Data
KDs (nM)
Conclusions Under some conditions, ?70 binds
tighter to ?cc than to core! Binding of ?32
to ?cc is much weaker than to core
53
Dissecting the Interactions

• The total binding energy that results in a KD is
  a sum of the energy gained by binding events
  minus the energy needed to make conformational
  changes.
• To fully dissect the importance of different
  interactions, we need to account for the
  conformational changes.

Gibbs free energy ?G ?H-T?S -RTlnKD
-2.3RTlogKD ?Gcore ?G?cc ?G?flap -
?Gconformation Model ?70 binds to ?cc, opens
up and then binds to ?-flap. This is essentially
iso-energetic, but unmasks promoter recognition
sites in regions 2.4 and 4.2
54
How can sigma70 bind to bcc almost as tightly as
to core when other sigma-core interactions are
also important?

• If the binding of sigma70 to core is about 3-4
  times tighter than to bcc, how can the region 4
  - b flap tip helix interaction be of significant
  strength?
• (10-8) x (10-6) 10-14? no, 10-8-10-9 M
• Suggestion
• 1. Initial interaction of sigma70 is with bcc.
  
• 2. Energy is required to open up sigma so that
  it can make additional interactions with core.
• 3. The energy required to open is almost equal
  to energy gained by additional interactions

55
4 Ways to Exchange Sigma

• General steady-state estimates
• 1000 molecules core/cell
• mM concentration of core in cell
• 500 core/cell actively transcribing at any time
• Models for where free core comes from
• Excess of core over total sigmas, available for
  sigma binding
• Free core is released after termination of
  transcription and can bind a sigma factor.
• During steady-state 400-500 core/min are
  released.
• Newly synthesized core is able to bind a sigma
  factor
• During steady-state 25 core/min are made (for
  40 min doubling time).
• A sigma factor dissociates from a holoenzyme and
  a new sigma factor can bind.
• Expect very little free core due to high protein
  concentrations in the cell compared to KDs and
  the fact that core may be limited.

56
Sigma Competition for Core in vivo?

• Holoenzyme populations change rapidly in vivo
• Changes in global transcription patterns can be
  seen soon after physiological shifts
• We see new RNA that is sigma32 dependent within 2
  min after new sigma32 is made
• We see that when sigma32 is overproduced in vivo,
  sigmaF-dependent transcription decreases
• (Kai Zhao, Liu and Burgess, JBC 2005) Kai
  Zhao, unpublished results)
• Models for sigma exchange
• Holoenzyme distribution is in rapid equilibrium
• But if binding constants and rates of
  dissociation measured in vitro are indicative of
  in vivo, then exchange may be too slow
• Exchange rates might by faster than expected
  in vivo due to different solution conditions or
  presence of small molecules or proteins that act
  as exchange factors
• Expect very little free core due to high
  protein concentrations in the cell compared to
  KDs and the fact that core may be limited.
• Competition is based on the active amount of each
  sigma and its relative binding constant for core
• Holoenzyme distribution is not in rapid
  equilibrium, only forms when sigmas bind to free
  core
• That is in excess over total sigma
• Released after termination of transcription
• Newly synthesized
• Competition is governed by relative rates of
  association of sigma for core and the relative
  abundance of each active sigma. Once bound,
  sigmas dont dissociate until they are released
  during transcription

57
Acknowledgments
Grant support from NIGMS
58
(No Transcript)
59
Summary of Mapping Results
Co- immobilization
Far-Western
700
900
1100
1300
100
500
300
- - - - /- - -
/- - - - -
'
?
A
B
C
E
F
G
D
H
? 178a.a.
E
F
G
B
C
D
H
? 220a.a.
B
C
D
E
F
G
H
? 260a.a.
B
C
D
E
F
G
H
? 270a.a.
G
C
D
E
F
H
? 280a.a.
C
D
E
F
G
H
? 290a.a.
G
C
D
E
F
H
1-280a.a.
A
B
1-300a.a.
A
B
1-309a.a.
A
B
33-309a.a.
A
B
60-309a.a.
A
B
100-309a.a.
B
B
150-309a.a.
200-309a.a.
B
260-309a.a.
60
Summary of Sigma70 Primary Binding Site on b
'
?
G
A
B
C
D
E
F
H
279
B
309
233
309
260
Interaction Domain
265 284 292 309
Helix
Helix
Predicted secondary structure
Predicted coiled coils
61
Possible coiled coil interactions
L
E
295K 302D 309D
R
M
A
Q275
A
R
L
L
V
N
R
R
V
L
g
g
c
c
L
d
L
d
f
N
E
D
D
N
R
f
a
a
b
Y
A269
G
e
b
e
N
V
R
R
D
L
K
L
N
R
S297
D266
N
Q
I
300E 293Q
D
R
K
E280
62
WT N266D Y269A R275Q K280E R293Q E295K R297
S Q300E A302D N309D
Far-Western with b1-319 mutations
A
Western
Far-Western
B
Relative sigma70 binding of mutants versus. WT
63
Screen of Tanaka-Marriott Library (Marine Sponge
Extracts)
D7
H4
G1
E12
E1
A7
B12
64
LRET Analysis of 4 Initial HTS Hits
65
Inhibition of in vitro Transcription
D7
66
Kinetic Assays
ka
kd dissociation rate ka association rate
At equilibrium kdholo kascore

67
Dissociation Rates
s70
s32
T1/2 (min) 29 28 23 19
T1/2 (min) 2 4 3 10
Starting Complex 10 nM Tb-core/ 40 nM F-s70 or
10 nM F-s32 Incubation Time 1 hour at 100 mM
NaCl and 22oC Competitor 100X s70 or 300X s32
68

• Tips/Precautions in LRET Binding Experiments
• Purify your derivatized protein from free dye.
  There is a very big problem with free dye causing
  diffusion-controlled LRET. Dialysis is not good
  enough! You must re-purify labeled protein.
• You can label either specifically at single
  sites, such as single cyteines, or randomly at
  lysines. The latter is much more convenient and
  yet works well on any pure protein.
• Validate your system Is all your
  derivatized protein active? (try native gel
  shifts). Are the binding properties of
  the labeled protein the same as the unlabeled
  protein? (do competition experiments). Is
  the binding reaction at equilibrium? (pre-form
  complex, add excess competitor, follow signal
  versus time). Are you in the correct assay
  window? (probably only good for Kd between
  10-10 to 10-6 M or even 10-9 to 10-7 M)

69
References on LRET, protein labeling, HTS,
binding constants V Bergendahl, L Anthony, T
Heyduk, and R Burgess, On-column
tris(2-carboxyethyl)phosphine reduction and
IC5-maleimide labeling during purification of
RpoC fragment on a nickel-nitriloacetic acid
column. Anal. Biochem. 307 368-74, 2002 V
Bergendahl, T Heyduk, R Burgess, LRET-based
high-throughput screening assay for inhibitors of
essential protein-protein interactions in
bacterial RNA polymerase. Appl. Environ.
Micro.691492, 2003 B Glaser, V Bergendahl, N
Thompson, B Olson, R Burgess, LRET-based
high-throughput screening of a small-compound
library for inhibitors of bacterial RNA
polymerase. ASSAY Drug Develop Technol 5759-768,
2007 B Glaser, V Bergendahl, L Anthony, B Olson,
R Burgess, LRET-based measurements of sigma
factor-core RNA polymerase binding constants in a
homogeneous assay. PLOS One, in press, 2009
70
Dissociation Rates
Starting Complex 10 nM Tb-core/ 40 nM
F-s70 Unlabeled competitor 4000 nM ?70 Holo
incubation time 1 hour 100 mM NaCl
Non-dissociable holo
71
Computer Simulation of Sigma-Core Binding Kinetics
Add 5 nM core, 2 nM ?70 5 nM ?32. At 60 sec,
add 10 nM ?70
Undergrad Chem E Chris Hacker, with recent help
from Beth and Rob Chumanov
72
Competition of Sigma Factors for Core RNAP

• Holoenzyme populations change rapidly in vivo
• Can occur in less than 2 minutes
• Is the competition of sigma factors for binding
  to core based on a rapid equilibrium?
• Concentrations of core RNAP and s are very high
  in the cell (mM).
• KD of interaction is very tight (nM),
  dissociation half-lives are very slow (3-30 min).
• It is difficult to imagine much free core RNAP
  in the cell due to holoenzyme dissociation.
• Our data indicate that competition for s binding
  to core RNAP primarily occurs when core becomes
  free after transcription or when the proteins are
  newly synthesized.
• This competition would be regulated by the rates
  of association and concentration of the proteins.
• However, cellular components or conditions in
  the cell could work to alter the levels of the
  proteins or to change the kinetics.

73
Non-dissociable Holo
100 mM NaCl
500 mM NaCl
Starting Complex 10 nM Tb-core/ 40 nM F-s70 or
10 nM F-s32 Competitor 100X s70 or 300X
s32 Similar results were observed with Kglu
Caution We dont yet understand why this
happens, but it suggests that the properties of
holo may vary depending on how long since the
holo formed
74
Sigma70-?coiled-coil Interaction
75
4 Ways to Exchange Sigma

• General steady-state estimates
• 1000 molecules core/cell
• mM concentration of core in cell
• 500 core/cell actively transcribing at any time
• Models for where free core comes from
• Excess of core over total sigmas, available for
  sigma binding
• Free core is released after termination of
  transcription and can bind a sigma factor.
• During steady-state 400-500 core/min are
  released.
• Newly synthesized core is able to bind a sigma
  factor
• During steady-state 25 core/min are made (for
  40 min doubling time).
• A sigma factor dissociates from a holoenzyme and
  a new sigma factor can bind.
• Expect very little free core due to high protein
  concentrations in the cell compared to KDs and
  the fact that core may be limited.

76
Fitting the binding data to get binding constants
77
Beta Epitope Mapping
C-His ?
N-His ?
C-His ?
N-His ?
C-terminal MAb
N-terminal MAb
Novagen
Hyd
Hyd
NTCB
Markers
NTCB

150
100
75
50
35
25
15
78
Equilibrium dissociation binding constants for
sigma-core binding
10 nM core (fixed)
79
Native Gel Shift Assay
s70
Fs (780 nM) Tb-core or core (90-900
nM) Incubated 1 hour at 22?C Run 4 hours at
4?C 4-12 Bis-Tris gel
s32
80
Altering the Sigma Exchange Rate?

• Anti-sigma factors
• AsiA - Anti-sigma70 in T4 infected cells
• RsdA - Anti-sigma70
• FlgM Anti-sigmaF
• DnaK Anti-sigma32
• RseA Anti-sigmaE
• FecR Anti-sigmaFecI
• Cellular molecules
• ppGpp, DksA, DNA, and small RNAs, metabolites?
• Environmental conditions
• pH, salt, temperature

81
DksA and ppGpp

• ppGpp regulates promoter usage during amino acid
  depletion.
• ppGpp alone does not affect transcription in
  vitro as much as in vivo.

• DksA is a 17 kDa protein that increases the
  inhibition of certain promoters by ppGpp, in
  vitro.
• DksA is abundant in the cell (30,000
  copies/cell). (Rutherford and Gourse)
• DksA binds core and holo.

Hypothesis DksA and ppGpp may play a role in
speeding the rate of exchange of sigmas for core
Paul et al., Cell (2004)
82
Dissociation of Holo (10 nM Tb-core/50 nM
Alexa555 Sigma70) by ppGpp and DksA at 26oC, 150
mM KGlu
Conclusion ppGpp and/or DksA dont affect rate
of sigma70 dissociation from core
83
Does DNA Alter the Sigma-Core Interaction?

• Hypothesis Non-specific DNA may act to speed
  the exchange rate of sigma factors bound to core.
• E ? E?
• Dns Dns
• E-Dns E?-Dns
• (E core E?holo, Dns non-specific
  DNA)

84
Effect of DNA on Dissociation of Holo (10 nM
Tb-core/50 nM Alexa555 Sigma70) by Competitors
at 26oC, 150 mM KGlu
Conclusion non-specific DNA doesnt affect rate
of sigma70 dissociation from core
85
Association kinetics of 10 nM Tb-core/50 nM
Alexa555 Sigma70) in presence of both DksA and
ppGpp at 37oC, 150 mM KGlu
Conclusion ppGpp and/or DksA dont affect rate
of sigma70 binding to core
86
SigmaS

• SigmaS acts differently than sigma70 in Kglu.
• Binding strength seems to increase at higher
  concentrations.
• This may be physiologically relevant.
• Binding constants will be done at multiple
  concentrations to see if the strength increases
  with salt concentration.

40 nM IC3-SigS 20 nM Tb-core
40 nM IC3-SigS 20 nM Tb-core
87
Sigma70

• The difference between NaCl and Kglu is quite
  dramatic. This is representative of sigmaE, F,
  54, and FecI.
• 150 mM Kglu will be used in binding constant
  assays because it is close to physiological
  levels.

(Bryan Glaser)
88
Sigma32

• Sigma32 binding to core is not as dependent on
  salt type or concentration.

40 nM IC3-Sig32 20 nM Tb-core
40 nM IC3-Sig32 20 nM Tb-core
89
(Veit Bergendahl)
90
Interaction of sigmas with core - role of
bcoiled coil in overall binding

• Ratio of KD for sigma binding to core versus bcc
• Sigma KD bcc/KD core
• sigma70 3.3
• sigma32 25
• 150 mM KGlu, 50 mM Tris pH 7.9, 5 glycerol,
  26oC
• These values are consistent with results of
  far-Western blotting analysis and complex
  formation between these sigmas and bcc by native
  gel shift

91
Sigma32 and Sigma70 differ in how they bind to
core

• Unlike sigma70, sigma32 is not released from core
  when its holoenzyme binds to phosphocellulose or
  BioRex70
• Sigma32 seems to derive more of its binding
  energy from interactions other than the
  interaction with the bcc
• 3. Sigma32 binding to core is not sensitive
  to NaCl concentration
• 4. At very low salt (lt50 mM), no drop off of
  sigma32 binding to core is seen (maybe sigma32
  can bind to core dimer, and sigma70 cant)

92
Prepare Labeled Protein

• Overproduce proteins in E. coli and purify
  protein
• Protein is eluted in carbonate buffer (100 mM
  NaHCO3 and NaCl, pH 8.3)
• Label lysines with 5-fold molar excess of dye
• Pass over DE52 column to remove unreacted dye
• Purify on size exclusion column to separate free
  dye from protein
• Dialyze into storage buffer (150 mM Kglu, 50 mM
  Tris HCl pH 7.9, 0.1 mM EDTA, 0.1 mM DTT, 50
  glycerol)

Elution Fractions
Start
A
B
C
MW
C
Sigma54
B
A
Free Dye
93
Purified and Labeled Sigma Factors
Coomassie Stain
FecI E F
32 S 54 70
94
References on LRET, protein labeling, HTS,
binding constants V Bergendahl, L Anthony, T
Heyduk, and R Burgess, On-column
tris(2-carboxyethyl)phosphine reduction and
IC5-maleimide labeling during purification of
RpoC fragment on a nickel-nitriloacetic acid
column. Anal. Biochem. 307 368-74, 2002 V
Bergendahl, T Heyduk, R Burgess, LRET-based
high-throughput screening assay for inhibitors of
essential protein-protein interactions in
bacterial RNA polymerase. Appl. Environ.
Micro.691492, 2003 V Bergendahl, B Glaser, L
Anthony, R Burgess, LRET-based measurements of
sigma factor-core RNA polymerase binding
constants in a homogeneous assay. in preparation,
2004
95
Sigma Competition with Labelled s70 for
b-Fragment Binding
96
Fitting the binding data to get binding constants