From the Unité de Physico-Chimie des Macromolécules Biologiques, CNRS URA 1773, Département de Biologie Moléculaire, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France
Received for publication, November 6, 2000, and in revised form, February 9, 2001
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ABSTRACT |
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During infection of Escherichia coli,
the phage T4 early protein AsiA inhibits open complex formation by the
RNA polymerase holoenzyme E In Escherichia coli RNA polymerase, the core
enzyme E (subunit composition Because it is a coactivator of transcription from T4 middle promoters
and, simultaneously, a transcription inhibitor of bacterial or T4 early
promoters (3), AsiA might also be able to cause the rapid arrest of
transcription from early promoters, concomitant with the start of
MotA-dependent middle transcription. Assigning this
function is a long-standing and unresolved question in phage T4 biology
(10). It has been recently shown, however, that this transcription
shutoff also occurs in the absence of AsiA (11), although the same
study confirmed that transcription of E. coli genes is
rapidly and strongly inhibited in vivo when AsiA is overproduced.
In vitro transcription studies have helped to outline the
mechanism of inhibition by AsiA. This 10-kDa protein binds to region 4.2 of Materials--
High Pure spin columns and the Pwo
polymerase were from Roche Molecular Biochemicals (Mannheim, Germany).
T4 polynucleotide kinase was purchased from New England Biolabs Inc.,
and DNase I was from Worthington. Magnetic DynaBeads were from Dynal,
Inc., and the 8-25% polyacrylamide PhastGels were from Amersham
Pharmacia Biotech.
Plasmids and DNA Fragments--
A 207-bp lacUV5 DNA
fragment (20) was inserted at the EcoRI site of plasmid
pJCD0 (21) upstream of the rrnB T1 and T2 terminators to
yield plasmid pGO1. A 665-bp lacUV5 fragment comprising the
two terminators was isolated by polymerase chain reaction with template
pGO1 and the following primers: 5'-CGCCAGGGTTTTCCCAGTCACGAC and
5'-GGATTTGTCCTACTCAGGAG. A 220-bp lacUV5 fragment was
isolated by polymerase chain reaction using plasmid
pBR-lacUV5 as a template (20) together with primers A1
(5'-GGCGTATCACGAGGCCCTTTCG) and B1 (5'-GCTGGCACGACAGGTTTCCCGA). Primer
A1 was end-labeled with T4 polynucleotide kinase and
[ Purified Proteins and Standard Reaction Conditions--
E.
coli RNA polymerase holoenzyme was purified according to Burgess
and Jendrisak (22) as modified by Marschall et al.
(21). AsiA protein from phage T4 was purified as previously described (16). Core RNA polymerase was prepared according to Lederer et
al. (23), and Isolation on Magnetic Beads of the Ternary RNA
Polymerase·AsiA·lacUV5 Complex--
The biotinylated
lacUV5 DNA fragment was prepared as described above. After
purification, the biotinylated fragment was immobilized on Dynal
streptavidin magnetic beads (12). RNA polymerase holoenzyme (40 pmol)
and an 8-fold molar excess of AsiA were incubated in a 30-µl reaction
for 30 min at 37 °C in buffer A containing 100 mM KGlu
and 0.125% (v/v) Tween instead of bovine serum albumin. The binding
reaction was then added to the immobilized lacUV5 fragment,
and the mixture was incubated for 60 min at room temperature. The beads
were collected by centrifugation, and the supernatant containing the
unbound proteins was withdrawn (see Fig. 2, lane 2). The
beads were then washed twice with KGlu buffer, and the bound proteins
were eluted after a 1-h incubation at room temperature in 1% SDS (see
Fig. 2, lane 3). The samples and controls were analyzed by
electrophoresis under denaturing conditions on an 8-25% (w/v)
polyacrylamide gradient PhastGel.
Single Round Transcription Assays--
Prior to assembly for
transcription reactions, RNA polymerase, AsiA, and the
lacUV5 template were separately diluted on ice in buffer A
containing either 100 mM KCl or the indicated KGlu concentration (100-400 mM). Runoff transcription reactions
were carried out at 37 °C under the following conditions: 30 nM RNA polymerase holoenzyme, 2 nM
lacUV5 template, the indicated molar excess of AsiA over the
polymerase concentration, 100 µM ATP, 100 µM CTP, 100 µM GTP, 10 µM
UTP, 0.5 µCi of [ Abortive Transcription--
Abortive transcription reactions
(26, 27) were carried out at 37 °C in buffer A containing the
indicated KGlu concentrations and the final concentrations of the
following components: 500 µM ApA, 50 µM
UTP, 2 nM lacUV5 DNA fragment, and 0.5 µCi of
[ Gel Retardation Assays--
Stock solutions of AsiA and RNA
polymerase were prepared on ice in buffer A with 100 mM KCl
or KGlu. When present, AsiA was in 6-fold molar excess relative to the
highest polymerase concentration used. Incubations were at 37 °C.
RNA polymerase·AsiA complexes were first formed during 15 min, and
the enzymes were then incubated for 80-90 min with the radioactively
labeled lacUV5 fragment at 0.02-0.065 nM. After
addition of heparin (55 µg/ml), the samples were loaded onto 5%
native polyacrylamide gels prepared in Tris borate/EDTA buffer and
electrophoresed at 120 V at room temperature.
DNase I Footprinting--
Complexes with the labeled
lacUV5 promoter (at a 3 nM final concentration)
were formed during 50 min at 37 °C in buffer A containing 200 mM KGlu or 100 mM KCl, using a 6-fold molar
excess of AsiA or the cAMP·CAP complex as indicated in Fig. 7, with
purified RNA polymerase or reconstituted Single Round Transcription Analysis in KGlu
Buffers--
Transcription inhibition by AsiA of E Existence of a Ternary Complex Formed at the lacUV5 Promoter by
E KGlu Increases the Affinity of the Holoenzyme·AsiA Complex for
the lacUV5 Promoter--
Given the possibility of forming in KGlu
buffers a ternary complex between RNA polymerase, AsiA, and
lacUV5, we used gel shift assays to determine the effect of
AsiA on holoenzyme affinity for the lacUV5 promoter. We
measured the formation of heparin-resistant complexes in KGlu and KCl
buffers in the absence and presence of AsiA. Fig.
3A shows a gel retardation
assay of complexes formed in 100 mM KGlu, and Fig.
3B shows an analogous experiment performed in the presence
of 100 mM KCl. In either buffer, a clear retarded complex
was observed at all holoenzyme concentrations. After quantification by
phosphorimaging, the gel shift data were fitted to the equation of a
rectangular hyperbola using a nonlinear regression program. The results
can be summarized as follows. In KGlu buffer, even in the presence of
AsiA, a fractional saturation value close to 1 was observed at high
holoenzyme concentrations. In 100 mM KGlu, an apparent
dissociation constant (KD) of 0.35 ± 0.07 nM was found for E KMnO4 Reactivity of the
E Kinetic Analysis of Open Complex Formation by the Holoenzyme·AsiA
Species--
The process studied above by KMnO4 reactivity could also
be monitored using an abortive initiation assay that probes only the
kinetically competent species. By this method, we analyzed the kinetics
of binding of the holoenzyme to the promoter without and with AsiA
previously bound to the enzyme. When the reaction was initiated by
addition of E
Transcription by E The Ternary Complex E Role of Upstream Contacts in the Properties of the Ternary
Complex--
We used reconstituted holoenzymes to compare the behavior
of normal E
To test this possibility, we used the reconstituted holoenzymes
E
In KGlu, we looked further for the presence of AsiA in the footprint
when the cAMP·CAP complex was used to activate the system. As in the
pattern afforded by the ternary complex, bands at position In this study, we describe the behavior of the
E AsiA inhibits open complex formation by binding directly to the
conserved region 4.2 of the By analyzing the mode of action of AsiA in the presence of the
glutamate anion, we show that this mutual exclusion can be by-passed.
As for many other DNA/protein systems in which Cl The transcriptional activity of the ternary complex strongly implied
the existence of a compensatory mechanism. It is well documented that
the The compensatory mechanism documented here at lacUV5 has
also been observed, almost unmodified, on lacPS,
a parent and weaker promoter in which the At this point, we will briefly consider the transcription properties of
the E In this view, this study demonstrates that promoter upstream contacts
mediated by 70 at
10/
35 bacterial
promoters through binding to region 4.2 of the
70
subunit. We used the
10/
35 lacUV5 promoter to study the
properties of the E
70·AsiA complex in the presence of
the glutamate anion. Under these experimental conditions, inhibition by
AsiA was significantly decreased. KMnO4 probing showed that
the observed residual transcriptional activity was due to the slow
transformation of the ternary complex E
70·AsiA·lacUV5 into an open
complex. In agreement with this observation, affinity of the
enzyme for the promoter was 10-fold lower in the ternary complex than
in the binary complex E
70·lacUV5. A tau
plot analysis of abortive transcription reactions showed that AsiA
binding to E
70 resulted in a 120-fold decrease in the
second-order on-rate constant of the reaction of E
70
with lacUV5 and a 55-fold decrease in the rate constant of
the isomerization step leading to the open complex. This ternary
complex still responded to activation by the cAMP·catabolite
activator protein complex. We show that compensatory
E
70/promoter upstream contacts involving the C-terminal
domains of
subunits in E
70 become essential for the
binding of E
70·AsiA to the lacUV5 promoter.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
'
) associates
with the
subunit to form the holoenzyme E
, the species able to
initiate transcription at specific promoter sites on bacterial or phage
genomes. The nature and properties of the
subunit present in a
holoenzyme at a given promoter, together with the ability of the
-carboxyl-terminal domain
(
-CTD)1 to recognize an
upstream element of the promoter, determine whether and how often
transcription will start at this site (1). These principles are nicely
illustrated by the regulation of transcription during the development
of phage T4 in E. coli. Here, all the phage genes are
transcribed by the host RNA polymerase, the structure and
molecular properties of which are modified by phage-coded proteins,
resulting in the sequential utilization of early, middle, and late
promoters (2, 3). Immediately after infection, T4 early promoters, with
their bacterium-like
10 and
35 recognition sequences, are
transcribed by E
70, the host holoenzyme harboring
70, the major host
factor. T4 middle promoters
contain a
10 element closely matching the
10 consensus sequence for
70, but the
35 element is replaced by a so-called MotA
box, a
30 binding site for the phage-coded middle transcriptional
activator MotA (4, 5). In addition to E
70 and MotA,
middle mode transcription also requires the presence of another phage
early protein, the anti-
factor AsiA (6). Upon binding the MotA box,
MotA protein activates recognition of middle promoters by a holoenzyme
E
70·AsiA complex (7, 8). Here, AsiA appears to act as
a molecular device that switches
70 from the early to
the middle class of T4 promoters. Transcription at late promoters is
closely coupled with T4 DNA replication and requires a novel T4-encoded
subunit, gp55 (9).
70. In the E
70·AsiA complex,
this binding blocks the normal interaction between
70
and the
35 upstream promoter element (12-14). Although AsiA strongly inhibits open promoter complex formation and transcription by E
70 from a
10/
35 promoter like lacUV5 or
the T4 early promoter P15.0, the holoenzyme is resistant to AsiA
inhibition at promoters that, like galP1, lack a
35
consensus motif and contain an "extended
10" motif (12, 13, 15,
16). These observations led to a simple model in which the interactions
of domain 4.2 in
70 with the
35 promoter element or
with AsiA are mutually exclusive. This model explains the effect of
AsiA on E
70 at a
10/
35 promoter under classical
assay conditions (13). However, at lacUV5, these
experimental conditions preclude a detailed analysis of the repression
mechanism. In this study, we have looked for conditions allowing the
maintenance of residual transcriptional activity. For this purpose, we
selected a salt that enhances the interactions between the RNA
polymerase partners. Replacing the chloride anion by glutamate
increases the affinity of RNA polymerase for its promoters (17, 18).
Such a change is also likely to improve the interaction between protein
partners (19). We expected, and indeed observed, that in the presence
of the Glu
anion, E
70 could still form a
kinetically competent complex in the presence of AsiA. This allowed us
to quantify free energy changes occurring in the overall reaction and
to identify specific compensatory interactions between the holoenzyme
and the promoter that permit RNA polymerase to overcome the otherwise
strong inhibition brought about by AsiA.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (3000 Ci/mmol) or used with unlabeled primer
B1 in a polymerase chain reaction to prepare the 220-bp
lacUV5 fragment labeled at the 5'-end on the non-template
strand. This fragment was used in the gel shift assay and the DNase I
footprinting experiments. Primer A1 biotinylated at the 5'-end was used
in a polymerase chain reaction to obtain the biotinylated 220-bp
lacUV5 fragment.
70 was obtained using the
overproducing strain M5219/pMRG8 and the published purification
procedure (24). Catabolite activator protein (CAP) was prepared as
described by Ghosaini et al. (25), and the
-235 RNA
polymerase was a gift from Dr. Evelyne Richet. The experiments
described below were performed in buffer A containing 40 mM
Hepes (pH 8.0), 10 mM MgCl2, 500 µg/ml bovine
serum albumin, 1 mM dithiothreitol, and either 100 mM KCl or the indicated KGlu concentration.
-32P]UTP, and 250 µg/ml heparin.
RNA polymerase and AsiA were first mixed at 37 °C for 15 min.
Template was added to allow open complex formation for 30 min, followed
by addition of the nucleotides and heparin. Elongation reactions lasted
10 min and were stopped by mixing equal volumes of reaction mixtures
with 20 mM EDTA in xylene cyanol-containing 95% formamide.
Following heating at 65 °C, the samples were electrophoresed on a
7% polyacrylamide sequencing gel, and the transcripts were quantified
with a PhosphorImager.
-32P]UTP. The lag assay in Fig. 5A was
performed with 30 nM RNA polymerase (final concentration),
previously incubated for 15 min either with buffer or with an 8-fold
molar excess of AsiA. The experiments with reconstituted RNA
polymerases were performed using a fixed time assay at a 100 nM final concentration of the different enzymes. Tau plot
analysis (26, 28) was carried out to measure the average time
obs required for open complex formation at
lacUV5 with and without AsiA previously bound to the
holoenzyme. For each reaction, the amount of UTP incorporated was
plotted versus time. The two parameters
obs
(minutes) and final steady-state velocity V (mole of product
ApApUpU/mol of promoter/min) were determined using a Kaleidagraph
program that performed a least-squares fit of the data to the following
equation: Y = V·t
V·
obs(1
exp(t/
obs)), where Y is the amount
of product, and t is time in minutes (28).
-235 RNA polymerase
(each at a 100 nM final concentration). Complexes were then
treated with DNase I (at a 80 ng/ml final concentration) for 30 s
(or 15 s in the absence of RNA polymerase). Protected bands were
identified on the pattern afforded by the migration of the same
fragment treated for the G + A sequencing reaction (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70,
the RNA polymerase holoenzyme, has been conveniently analyzed with well
defined DNA templates (13, 15, 16). Here, we performed single round transcriptions with a DNA linear fragment containing the
lacUV5 promoter upstream of a strong transcriptional
terminator and generating a 178-nucleotide transcript. This construct
was used to assess the effect of increasing AsiA concentrations
(relative to holoenzyme) on runoff transcription reactions when the
assays were performed in KGlu buffers of increasing ionic strength as
compared with KCl, after a 30-min incubation time with the promoter in
each case. In the absence of AsiA, the effect of the KGlu buffers
(100-400 mM KGlu) was a 25-30% decrease in
transcriptional activity relative to that measured in 100 mM KCl, an effect that was ionic strength-independent. Fig.
1 shows that the nature and concentration
of the buffer both have a marked effect on the extent of transcription
inhibition by AsiA. Strong inhibition was observed in 100 mM KCl and more so in 400 mM KGlu. In contrast,
at all other KGlu concentrations (100-300 mM),
transcription was significantly less inhibited, and the corresponding
residual activities reached a 75-80% plateau as the AsiA
concentration was raised. We therefore utilized the 100-300
mM KGlu concentration range to investigate in greater detail the behavior of the enzyme·inhibitor complex, the
E
70·AsiA entity, relative to the lacUV5
promoter.
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Fig. 1.
Effect of reaction conditions on AsiA
inhibition of single round transcription by holoenzyme
E 70 from
lacUV5. Runoff transcription reactions were
carried out as described under "Experimental Procedures." The graph
shows the percent activity relative to that of controls in the absence
of AsiA. The reactions were run with 30 nM
E
70 and 2 nM lacUV5 DNA fragment
in buffer A containing the following salts: 100 mM KCl
(
) and 100 mM (
), 200 mM (×), 300 mM (
), and 400 mM (+) KGlu.
70·AsiA--
The results described above prompted us
to check whether AsiA was present in a stable ternary complex formed
with E
70 and the promoter in the presence of 100-300
mM KGlu. For this experiment, we used a biotinylated
lacUV5 DNA fragment immobilized on streptavidin-agarose
magnetic beads. The proteins found to be bound to this DNA fragment
were recovered and analyzed on an SDS-polyacrylamide gel (12).
AsiA (in an 8-fold molar excess over enzyme) was first added to the
holoenzyme in 100 mM KGlu (Fig.
2, lane 1), and the mixture
was incubated with the lacUV5 fragment to allow open complex
formation. Following this treatment, the holoenzyme was eluted from the
beads (Fig. 2, lane 3). Based on the relative Coomassie Blue
staining intensities, this eluted fraction was found to contain AsiA in
a stoichiometric ratio relative to the
subunit present in this
holoenzyme preparation (Fig. 2, lane 5). As a control, the
same experiment was performed in the presence of 100 mM
KCl. In this case, in contrast to the result observed in KGlu, previous
incubation of E
70 with an 8-fold molar excess of AsiA
led to the elution from the immobilized promoter of an extremely low
quantity of E
70, containing no detectable amount of AsiA
(data not shown). Furthermore, in DNase footprinting experiments
performed in the presence of 100 mM KCl with
E
70·AsiA on the lacUV5 fragment, the only
species that could be detected was a residual binary complex,
E
70·lacUV5 (see Fig. 7B
below). Therefore, in the presence of KGlu, AsiA appeared
to take part in a stable ternary complex at the lacUV5 promoter.
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Fig. 2.
SDS-polyacrylamide gel analysis of the
ternary
E 70·AsiA·lacUV5
complex. E
70 and AsiA (8-fold molar excess)
were incubated in buffer A containing 100 mM KGlu with the
biotinylated lacUV5 fragment on magnetic beads as described
under "Experimental Procedures" (lane 1). Lane
2, supernatant containing the unbound material; lane 3,
fraction eluted from the beads with 1% SDS; lane 4, AsiA;
lane 5, purified E
70; lane 6,
molecular size markers (in kilodaltons). In lanes 3 and
5, the arrows indicate the
subunit present in
this RNA polymerase preparation that migrated just above AsiA.
70/promoter binding.
This value was >10-fold higher when AsiA was previously bound to the
holoenzyme (KD = 4.7 ± 1.6 nM). In
100 mM KCl and in the absence of AsiA, the binary complex
displayed a KD of 1 ± 0.1 nM,
indicative of weaker binding of the enzyme to the promoter. In the same
buffer and in the presence of 600 nM AsiA, the formation of
a retarded species was greatly affected as the enzyme concentration
increased. An apparent KD of 100 ± 10 nM was calculated for the enzyme from the gel shift assay.
Taken together, these results indicate a qualitative but clear-cut
trend: shifting from glutamate to chloride destabilizes the binary
complex by a factor of 2.8 in the absence of AsiA. In its presence, the
RNA polymerase affinity for the promoter is even more affected because
the apparent affinity drops by a factor of 22-25 under the conditions
tested. These figures illustrate the destabilizing character of the
Cl
buffers often used in vitro to study
systems involving DNA/protein interactions.
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Fig. 3.
A, 100 mM KGlu: gel shift
assay showing the effect of AsiA on holoenzyme binding to the
lacUV5 DNA fragment. Binding was carried out in
buffer A containing 100 mM KGlu. When present, AsiA was at
600 nM (final concentration). The retarded species are
heparin-resistant complexes. Lane 1, free DNA; lane
2, DNA and AsiA; lanes 3 and 8, 0.5 nM holoenzyme; lanes 4 and 9, 1 nM holoenzyme; lanes 5 and 10, 5 nM holoenzyme; lanes 6 and 11, 20 nM holoenzyme; lanes 7 and 12, 100 nM holoenzyme. B, 100 mM KCl: gel
shift assay showing the effect of AsiA on holoenzyme binding to the
lacUV5 DNA fragment. RNAP, RNA polymerase.
70·AsiA·lacUV5 Complex in Potassium
Glutamate--
Potassium permanganate has been used to probe exposed
pyrimidines in single-stranded DNA (30). KMnO4 footprinting
experiments were performed to confirm that the complex visualized in
the gel retardation experiments in the presence of AsiA was an open
complex. We first compared the KMnO4 reactivities of the
complexes formed in KGlu with and without AsiA. KMnO4
sensitivity was observed in the binary complex
E
70·lacUV5 at positions +2, +1,
8,
9,
and
11 on the template strand of lacUV5 (data not shown),
characteristic of the open complex formed by E
70 at this
promoter (30). When AsiA was previously bound to E
70,
the same positions were found to react with KMnO4, although more slowly. The time course of establishment of this process was
monitored. At zero time, E
70·AsiA was incubated in 100 mM KGlu with the labeled promoter fragment (50 nM E
70, 300 nM AsiA, and 2 nM DNA), and the change in KMnO4 reactivity was
measured as a function of time. The results were quantified by
comparison with the footprinting signal obtained with
E
70 after allowing 60 min for maximal open complex
formation. The kinetics observed with the ternary complex fit a single
exponential (time constant
close to 26 min), and the value measured
at 60 min was 85% of the control (Fig.
4). This kinetic profile clearly differs
from the fast opening of the promoter in the binary complex (Fig. 4,
see the control at 10 min). In the presence of KGlu, the
species E
70·AsiA is therefore able to form an open
complex at the lacUV5 promoter, with a time course that is
nevertheless significantly slower than for the uninhibited
holoenzyme.
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Fig. 4.
Time-dependent change in
KMnO4 reactivity of
E 70·AsiA·lacUV5
complexes in 100 mM KGlu buffer. At zero time,
E
70·AsiA was incubated with the labeled DNA fragment
at the following final concentrations: 50 nM
E
70, 300 nM AsiA, and 2 nM DNA.
The gel image data were obtained with a PhosphorImager and analyzed
using the ImageQuant program. On the gel image, at each time point, a
rectangle was drawn so as to include the five KMnO4
reactive positions (see "Results"). After background
correction, the percentage of open complex was obtained by dividing the
integrated intensity of each rectangle by the intensity measured with
the open complex E
70·lacUV5 formed after 60 min. In parallel, KMnO4 reactivity was also measured with
E
70·AsiA·lacUV5 formed in 100 mM KCl buffer and yielded a value of 20% open complex at
the 90-min time point of the experiment.
70·AsiA instead of E
70
alone (40 nM in each case), we systematically observed an
important increase in the latency time required to reach the
steady-state rate of oligonucleotide synthesis. This increase was
monitored at several KGlu concentrations (Fig.
5A). No lag could be easily measured with the holoenzyme alone. When AsiA was previously bound to
the holoenzyme, a marked and roughly constant latency time was observed
in the presence of 100-300 mM KGlu (Fig. 5A).
We chose then 200 mM as a convenient KGlu concentration and
performed a tau plot analysis to determine the [E
70]
dependence of the observed lag time
obs without and with
an 8-fold excess of AsiA. At excess RNA polymerase over promoter concentration, the tau plot analysis is based upon a simple two-step model (Equation 1) for open complex formation (31, 32),
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Fig. 5.
Abortive transcription reactions.
A, time course of open complex formation at the
lacUV5 promoter. Kinetics of association between
E 70 and lacUV5 without (closed
symbols) or with (open symbols) AsiA previously bound
to the holoenzyme were initiated by addition of E
70 (30 nM final concentration) to the assay mixture described
under "Experimental Procedures" and containing 100 mM
(circles), 200 mM (squares), or 300 mM (triangles) KGlu in buffer A. B,
tau plot for open complex formation at the lacUV5 promoter
in 200 mM KGlu-containing buffer A. The
obs
values are plotted as a function of the reciprocal of
[E
70], without (
) or with (
) AsiA (8-fold molar
excess) previously bound to the holoenzyme. RNAP, RNA
polymerase.
where R is E
(Eq. 1)
70, KB is the
equilibrium binding constant of R to the promoter P, and
kf is the isomerization first-order rate constant
(31). Utilizing Equation 1 in the present case is thus an attempt to
analyze the effect of binding AsiA to R in terms of this two-step
model. Fig. 5B shows the considerable effect induced by AsiA
upon the holoenzyme concentration dependence of the lag time
, and
Table I reports the kinetic
parameters derived from these measurements. A strong kinetic penalty
is brought about by the addition of AsiA. The overall second-order
association constant KBkf is
decreased by ~120-fold. In terms of the two-step model of Equation 1,
this effect is mainly expressed at the isomerization step of the
pathway. Relative to the situation encountered with the free enzyme R,
there is a 55-fold decrease in kf in the presence of
excess AsiA. Despite a very large uncertainty in the values of
KB (a ratio of 2 is observed between the average
KB constants), we can safely conclude that, under
these experimental conditions, AsiA is not a competitive inhibitor of
RNA polymerase binding to the lacUV5 promoter.
Kinetic parameters of the abortive initiation reaction at lacUV5 by
E70 and E
70·AsiA in the presence of 200 mM KGlu
70 from the galP1 promoter
or from a promoter sequence lacking a
35 hexamer has been shown to be
essentially insensitive to inhibition by AsiA (12, 13, 15). In
agreement with this, we did not observe the marked lags described above with lacUV5 when we used a consensus galP1
promoter that lacks a
35 consensus sequence (data not shown). Thus,
the increased lags at lacUV5 specifically reflect an
imperfect interaction between E
70·AsiA and this
promoter with its functional
35 hexamer. We therefore looked for
indications of modified interactions between the polymerase and the
upstream regions of the promoter that could account for the observed
transcriptional activity of E
70·AsiA at this
10/
35 promoter.
70·AsiA·lacUV5 Is
Susceptible to Activation by CAP--
At the lacUV5promoter, the
cAMP·CAP complex is known to bind a site centered at
61.5 and to
activate open complex formation (33). We have analyzed the effect of
the cAMP·CAP complex on the kinetics of open complex formation by
E
70·AsiA. Fig. 6 shows
that when cAMP·CAP was incubated with the promoter before addition of
30 nM E
70 and with an 8-fold molar excess of
AsiA, the observed lag time was reduced from 52 to 18 min; and
interestingly, the two kinetics reached the same final steady-state
rate after the lag period. The cAMP·CAP complex is therefore able to
activate the holoenzyme entity E
70·AsiA. We suspected
that this effect could be mediated through the formation of a
quaternary E
70·AsiA·lacUV5·CAP complex
(see below).
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Fig. 6.
Effect of CAP on the kinetics of open complex
formation at the lacUV5 promoter. The reactions
(in 200 mM KGlu in buffer A) were performed in the presence
( ) or absence (
) of CAP. When present during the assay, CAP was
first mixed with 3.125 nM lacUV5 DNA fragment
and 250 µM cAMP. AsiA was at an 8-fold molar excess
relative to the final concentration of E
70 (30 nM).
70 and of E
70(
), a
holoenzyme containing
subunits with deletions in their CTDs (34,
35). Using the abortive initiation assay, we first measured, in the
presence of 180 mM KGlu, the extent of inhibition caused by
AsiA (6-fold molar excess) for three different holoenzymes, each at a
100 nM final concentration: native E
70,
reconstituted E
70, and reconstituted
E
70(
). Each holoenzyme was incubated for 30 min
with lacUV5 prior to the assay. The native holoenzyme was
30% inhibited. Reconstituted E
70 was 60% inhibited,
whereas reconstituted E
70(
) was almost totally
inhibited (97%). An
-CTD deletion was therefore able to almost
totally suppress the partial insensitivity to AsiA conferred to
E
70 by the interactions enhanced by the presence of
glutamate. Thus, promoter upstream contacts involving the
-CTD
appear to be essential to counteract the inhibitory effect of AsiA
bound to
70. Upstream contacts between the
promoter and the C-terminal domain of an
subunit of
E
70 are known to be further stabilized by the cAMP·CAP
complex bound upstream (36).
70 and E
70(
) in DNase I
footprinting experiments to probe the structure of these complexes with
and without AsiA. The ternary complex formed by
E
70·AsiA showed an extended protection pattern (Fig.
7A, lane 3) with,
however, notable differences compared with that produced by
E
70 (lane 4). The most visible difference was
the strong hypersensitive bands around position
35 in the
E
70·AsiA pattern. This footprint also showed an
increased protection around positions
57 to
62 relative to that of
E
70 (Fig. 7A, compare lanes 3 and
4). Under similar conditions and in the presence of 100 mM KCl (Fig. 7B), the RNA polymerase footprint on the DNA fragment was barely detectable, and we were unable to
observe the presence of the hyperreactive band that we considered as
the signature of the ternary open complex (Fig. 7B,
lane 5).
View larger version (99K):
[in a new window]
Fig. 7.
A, 200 mM KGlu: DNase I
footprint analysis of the E 70·lacUV5 and
E
70(
)·lacUV5 complexes formed
in the absence or presence of AsiA. Native holoenzyme was used in
lanes 3-5 and 11-13, and reconstituted
holoenzyme carrying the
deletion at the CTD was used in
lanes 6-8. AsiA was either preincubated with each
holoenzyme (lanes 3, 6, and 11) or
added after open complex formation (lanes 5, 8,
and 13). Controls are shown in lane 2 (no
protein), lane 9 (AsiA alone), and lane 10 (cAMP·CAP complex binding to lacUV5). Lane 11 shows the simultaneous presence of AsiA and CAP in the open complex
formed with the native holoenzyme (compare with lane
12). B, 100 mM KCl: DNase I
footprint analysis of E
70·lacUV5 in the
absence or presence of AsiA. In the footprint shown lane 5,
the native holoenzyme was preincubated with AsiA. RNAP, RNA
polymerase.
35 were
strongly visible in presence of
E
70·AsiA·lacUV5 and CAP, strengthening
the hypothesis that a quaternary complex forms in the presence of
cAMP·CAP (Fig. 7A, lane 11). Also, addition of
AsiA to a preformed E
70·lacUV5 complex did
not yield the pattern characteristically perturbed by AsiA (Fig.
7A, lanes 5 and 13), confirming that
the inhibitor cannot bind to
70 once the holoenzyme has
already formed an open complex (13). Finally, when it was preincubated
with AsiA, E
70(
) failed to form any stable
complex, and the lacUV5 fragment (5'-labeled on the
non-template strand) yielded a pattern similar to that obtained with
naked DNA (Fig. 7A, compare lanes 2 and 6). These results confirmed the conclusion that, in the
presence of the glutamate anion, AsiA is part of a stable and
functional open complex formed by E
70 at the
lacUV5 promoter. Moreover, this particular open complex is
susceptible to activation by cAMP·CAP, as demonstrated by the DNase I
protection pattern showing evidence for the presence of the quaternary
complex E
70·AsiA·lacUV5·CAP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70·AsiA complex in the presence of KCl and KGlu, two
salts widely used in transcription studies. We have studied the
mechanism of inhibition exerted by AsiA when it is bound to
E
70 acting upon a
10/
35 promoter in
greater detail (13, 15, 16). By runoff transcription analysis on the
lacUV5 promoter, we show that the species
E
70·AsiA is much less inhibited in KGlu than in KCl
(Fig. 1). These observations suggested the existence of a
transcriptionally active ternary complex at lacUV5, a
typical bacterial promoter with
10 and
35 recognition sequences.
Active ternary complexes containing AsiA have been previously observed
at several promoters that do not require a
35 motif for open complex
formation (12, 13, 15). A number of convergent observations support the
hypothesis of a ternary complex at a
10/
35 promoter.
First, we showed that AsiA was present in a stoichiometric amount when
the E
70·AsiA entity was first incubated with
lacUV5 in KGlu (Fig. 2), but not in KCl. Thus, we ruled out
the possibility that, in the presence of KGlu, a spurious loss of AsiA
could explain the restoration of E
70 activity.
Furthermore, in Glu
buffer, only the extended DNase I
footprint formed by E
70·AsiA at lacUV5
possessed a distinctive AsiA "signature" in the
35 region (Fig.
7A, lanes 3 and 11). In contrast, with
AsiA bound to the holoenzyme in KCl, we observed only a weak protection
against DNase I, which we interpreted as due to residual amounts of the binary E
70·lacUV5 complex (Fig.
7B, lane 5). A third piece of evidence comes from
an investigation of the structure of the ternary complex by laser UV
photoreactivity.2 Laser UV
photo-footprinting of a RNA polymerase·promoter complex allows
precise probing of changes in the local structure of DNA (37). When we
formed an open complex at lacUV5 with E
70 in
200 mM KGlu, thymine dimer formation at positions
34 and
33 was suppressed, probably due to contacts between
70
and the
35 region (37). In contrast, no such effect was found when
AsiA was previously bound to E
70. However, in this case,
we observed signals in the
10 region, indicative of contacts that are
normally present in open complexes (38). This again strongly suggests
that even when access to open complex formation via the
35 region is
prevented by AsiA, RNA polymerase can nevertheless form a
transcriptionally competent species possessing the downstream
structural characteristics of a normal open complex. The presence of a
significant fraction of binary complex would have resulted in a
corresponding decrease in the
34 dimer signal. This was not observed.
We conclude that the effects we observed in the presence of glutamate
belong to a real and probably unique ternary complex referred to as
E
70·AsiA·lacUV5.
70 subunit. At a
10/
35 promoter, this binding interferes with the
interactions between
70 region 4.2 and the
35 motif
(12-14). Hindrance of these crucial interactions with concurrent
inhibition of open complex formation has been recently described in the
case of the gene 2 protein of bacteriophage T7 (39). Here, however, the
inhibitor gp2 binds to the
' subunit of E
70 and thus
indirectly disrupts the
35/
70 region 4.2 interaction
(39). Both inhibitors T4 AsiA and T7 gp2 are believed to operate in a
mutually exclusive mode: either the inhibitor is bound to the enzyme
with ensuing inhibition, or the polymerase is bound to the promoter
without inhibition. Moreover, in the latter case, the inhibitor can no
longer be bound to the enzyme (13, 39).
has
been replaced by Glu
, our results illustrate the general
observation that, in the presence of Glu
, there is a
substantial increase in the affinity of the proteins for their binding
sites on DNA (17, 40). The apparent KD values
determined by gel shift assay (see above and Fig. 3) are totally in
line with previous reports on the "glutamate effect" (40). Using
the abortive initiation assay and a simple two-state model (Equation 1)
for open complex formation, we showed by tau plot analysis that the
rate constant kf of the isomerization step was
decreased 55-fold when AsiA was present in the complex (Table I).
Inhibition by AsiA was therefore strongly maintained, as shown by the
global 120-fold effect on the second-order association constant
KBkf. In a parallel study, we
used the abortive initiation assay to assess the stability of the
ternary complex by heparin challenge (data not shown). The binary
complex (without AsiA) formed in 200 mM KGlu was absolutely
stable for >1 day. The ternary complex was much less stable and showed
a first-order decay with a half-life of ~18 h. This value is
considerably larger that the half-life associated with the conversion
of the closed to the open complex. The final ternary open complex
formed in KGlu is therefore clearly more stable than the closed intermediate.
subunit of RNA polymerase can also participate in promoter
recognition through specific interactions between
-CTD and upstream
regions of the
35 hexamer called "UP elements" (41-43). In
glutamate, we show here that the contribution to the binding energy
brought about by
-CTD binding to lacUV5 is
sufficient to partially overcome inhibition by AsiA and to allow the
formation of a ternary E
70·AsiA·lacUV5
active complex. Using the reconstituted holoenzyme E
70(
), we show that the deletion of the
-CTD
totally prevents the formation of the ternary complex. This complex
displays RNA polymerase/promoter upstream contacts as revealed by a
characteristic DNase I footprinting pattern that depends on
-CTD
binding upstream of position
40 (Fig. 7, lanes 3 and
11). A similar differential pattern was previously observed
in the absence of AsiA at promoters where the UP elements are essential
(34, 43). Also,
-CTD binding can be facilitated by the binding of
the cAMP·CAP complex at a site centered at
61.5. Interactions
between
-CTD and CAP allow one of the two
-CTDs to anchor more
tightly in the minor groove around position
43, upstream of the
35
hexamer (44). Interestingly, this interaction occurs regardless of
whether AsiA is bound to the adjacent
70 subunit, and it
allows CAP to activate the ternary complex
E
70·AsiA·lacUV5, thereby forming a
quaternary transcriptionally competent complex (Fig. 6).
10 region is altered, but
not the sequences participating in upstream contacts. Conversely, when,
at lacUV5, the UP element is reinforced by insertion of a
proper canonical sequence, the inhibition due to AsiA was found to be
already attenuated in the KCl-containing
buffer.3 It is therefore
likely that a better anchoring of the RNA polymerase·AsiA complex can
conceivably take place on the UP element at any promoter, but that a
clear balance favoring this repositioning might or might not require
the presence of potassium glutamate depending on the relative strengths
of the interactions at the
35 hexamer and at the UP element.
70·AsiA complex relative to phage T4 early and
MotA-dependent middle promoters. AsiA inhibits complex
formation at the T4 early promoter P15.0, possessing both a
35
recognition element and an extended
10 motif (16). When assayed in
KCl buffer, transcription from the T4 middle promoters
PuvsX, PrIIB1, PrIIB2, and P1 is strictly dependent on the
presence of both activators: MotA bound to the
30 middle promoter
sequence, and AsiA bound to
70 (8). Remarkably, when
assayed in KGlu, E
70 is able to transcribe correctly
from PuvsX (5, 7). This basal transcription by
E
70 at a middle promoter is not activated by MotA (45).
Furthermore, in KGlu, addition of AsiA to E
70 inhibits
both open complex formation at a PuvsX DNA fragment and
transcription from this middle promoter (7). Taken together, these
observations emphasize the point that AsiA acting alone behaves as a
repressor. It inhibits transcription from a MotA-dependent promoter in a manner reminiscent of its action upon a
10/
35 promoter. It is therefore likely that most of the RNA
polymerase/promoter contacts at
35 (and at
30 as well) are
perturbed by a local conformational change affecting
70
when it binds AsiA. This perturbation is, in turn, relieved by the new
contacts and therefore the new specificity conferred by the addition of MotA.
-CTD of E
70 are able to reduce the strong
structural and kinetic block brought about by AsiA. As a repressor,
AsiA changes radically the potential use of the enzyme modules involved
in the formation of contacts with the upstream region of the promoter.
We propose that in vivo, this drastic change also occurs.
Here, however, the
-CTDs are first ADP-ribosylated at the onset of
T4 infection (46). This modification plays a major role in regulating
promoter utilization since it irreversibly blocks the use of the
rescued pathway documented here. The efficiency of the
E
70·AsiA complex will now crucially depend on the
binding of MotA to those promoters containing the
30 middle promoter
motif (47, 48) and on the establishment of the proper contacts between the
70 subunit and MotA (49).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Evelyne Richet for the gift of
-235 RNA polymerase and Dr. Richard Ebright for providing the
UPprox-lacUV5 promoter fragment. We are grateful to Pascal
Roux and Cyril Badaut for help with the computer graphic programs. We
thank Dr. E. Richet for useful discussions and Dr. Tony Pugsley for
critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33-1-4568-8644;
Fax: 33-1-4061-3060; E-mail: akolb@pasteur.fr.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M010105200
2 G. Orsini and M. Buckle, unpublished results.
3 G. Orsini and A. Kolb, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
-CTD,
-carboxyl-terminal domain;
bp, base pair;
CAP, catabolite activator
protein;
KGlu, potassium glutamate.
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REFERENCES |
---|
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---|
1. | Gross, C. A., Chan, C., Dombroski, A., Gruber, T., Sharp, M., Tupy, J., and Young, B. (1998) Cold Spring Harbor Symp. Quant. Biol. 63, 141-155[Medline] [Order article via Infotrieve] |
2. | Kolesky, S., Ouhammouch, M., Brody, E. N., and Geiduschek, E. P. (1999) J. Mol. Biol. 291, 267-281[CrossRef][Medline] [Order article via Infotrieve] |
3. | Brody, E. N., Kassavetis, G. A., Ouhammouch, M., Sanders, G. M., Tinker, R. L., and Geiduschek, E. P. (1995) FEMS Microbiol. Lett. 128, 1-8[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Schmidt, R. P.,
and Kreuzer, K. N.
(1992)
J. Biol. Chem.
267,
11399-11407 |
5. |
Hinton, D. M.
(1991)
J. Biol. Chem.
266,
18034-18044 |
6. | Ouhammouch, M., Orsini, G., and Brody, E. N. (1994) J. Bacteriol 176, 3956-3965[Abstract] |
7. | Hinton, D. M., March-Amegadzie, R., Gerber, J. S., and Sharma, M. (1996) J. Mol. Biol. 256, 235-248[CrossRef][Medline] [Order article via Infotrieve] |
8. | Ouhammouch, M., Adelman, K., Harvey, S. R., Orsini, G., and Brody, E. N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1451-1455[Abstract] |
9. | Fu, T. J., Sanders, G. M., O'Donnell, M., and Geiduschek, E. P. (1996) EMBO J. 15, 4414-4422[Abstract] |
10. | Alberts, B. M. (1994) in Molecular Biology of Bacteriophage T4 (Karam, J. D., ed) , pp. 487-488, American Society for Microbiolgy, Washington, D. C. |
11. | Pene, C., and Uzan, M. (2000) Mol. Microbiol. 35, 1180-1191[CrossRef][Medline] [Order article via Infotrieve] |
12. | Colland, F., Orsini, G., Brody, E. N., Buc, H., and Kolb, A. (1998) Mol. Microbiol. 27, 819-829[CrossRef][Medline] [Order article via Infotrieve] |
13. | Severinova, E., Severinov, K., and Darst, S. A. (1998) J. Mol. Biol. 279, 9-18[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Sharma, U. K.,
Ravishankar, S.,
Shandil, R. K.,
Praveen, P. V.,
and Balganesh, T. S.
(1999)
J. Bacteriol
181,
5855-5859 |
15. | Pahari, S., and Chatterji, D. (1997) FEBS Lett. 411, 60-62[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Adelman, K.,
Orsini, G.,
Kolb, A.,
Graziani, L.,
and Brody, E. N.
(1997)
J. Biol. Chem.
272,
27435-27443 |
17. | Record, M. T., Jr., Courtenay, E. S., Cayley, S., and Guttman, H. J. (1998) Trends Biochem. Sci. 23, 190-194[CrossRef][Medline] [Order article via Infotrieve] |
18. | Leirmo, S., Harrison, C., Cayley, D. S., Burgess, R. R., and Record, M. T., Jr. (1987) Biochemistry 26, 2095-2101[Medline] [Order article via Infotrieve] |
19. |
Arakawa, T.,
and Timasheff, S. N.
(1984)
J. Biol. Chem.
259,
4979-4986 |
20. | Schaeffer, F., Kolb, A., and Buc, H. (1982) EMBO J. 1, 99-105[Medline] [Order article via Infotrieve] |
21. | Marschall, C., Labrousse, V., Kreimer, M., Weichart, D., Kolb, A., and Hengge-Aronis, R. (1998) J. Mol. Biol. 276, 339-353[CrossRef][Medline] [Order article via Infotrieve] |
22. | Burgess, R. R., and Jendrisak, J. J. (1975) Biochemistry 14, 4634-4638[Medline] [Order article via Infotrieve] |
23. | Lederer, H., Mortensen, K., May, R. P., Baer, G., Crespi, H. L., Dersch, D., and Heumann, H. (1991) J. Mol. Biol. 219, 747-755[Medline] [Order article via Infotrieve] |
24. | Gribskov, M., and Burgess, R. R. (1983) Gene (Amst.) 26, 109-118[CrossRef][Medline] [Order article via Infotrieve] |
25. | Ghosaini, L. R., Brown, A. M., and Sturtevant, J. M. (1988) Biochemistry 27, 5257-5261[Medline] [Order article via Infotrieve] |
26. | Buc, H., and McClure, W. R. (1985) Biochemistry 24, 2712-2723[Medline] [Order article via Infotrieve] |
27. | Busby, S., Kolb, A., and Minchin, S. (1994) Methods Mol. Biol. 30, 397-411[Medline] [Order article via Infotrieve] |
28. | Hawley, D. K., and McClure, W. R. (1982) J. Mol. Biol. 157, 493-525[Medline] [Order article via Infotrieve] |
29. | Maxam, A. M., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 560-564[Abstract] |
30. |
Sasse-Dwight, S.,
and Gralla, J. D.
(1989)
J. Biol. Chem.
264,
8074-8081 |
31. | McClure, W. R. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5634-5638[Abstract] |
32. | Chamberlin, M. J. (1974) Annu. Rev. Biochem. 43, 721-775[CrossRef][Medline] [Order article via Infotrieve] |
33. | Malan, T. P., and McClure, W. R. (1984) Cell 39, 173-180[Medline] [Order article via Infotrieve] |
34. | Kolb, A., Igarashi, K., Ishihama, A., Lavigne, M., Buckle, M., and Buc, H. (1993) Nucleic Acids Res. 21, 319-326[Abstract] |
35. | Igarashi, K., and Ishihama, A. (1991) Cell 65, 1015-1022[Medline] [Order article via Infotrieve] |
36. | Busby, S., and Ebright, R. H. (1994) Cell 79, 743-746[Medline] [Order article via Infotrieve] |
37. | Buckle, M., Geiselmann, J., Kolb, A., and Buc, H. (1991) Nucleic Acids Res. 19, 833-840[Abstract] |
38. | Buckle, M., Pemberton, I. K., Jacquet, M. A., and Buc, H. (1999) J. Mol. Biol. 285, 955-964[CrossRef][Medline] [Order article via Infotrieve] |
39. | Nechaev, S., and Severinov, K. (1999) J. Mol. Biol. 289, 815-826[CrossRef][Medline] [Order article via Infotrieve] |
40. | Ha, J. H., Capp, M. W., Hohenwalter, M. D., Baskerville, M., and Record, M. T., Jr. (1992) J. Mol. Biol. 228, 252-264[Medline] [Order article via Infotrieve] |
41. | Gaal, T., Ross, W., Blatter, E. E., Tang, H., Jia, X., Krishnan, V. V., Assa-Munt, N., Ebright, R. H., and Gourse, R. L. (1996) Genes Dev. 10, 16-26[Abstract] |
42. | Noel, R. J., Jr., and Reznikoff, W. S. (1998) J. Mol. Biol. 282, 495-504[CrossRef][Medline] [Order article via Infotrieve] |
43. |
Ross, W.,
Aiyar, S. E.,
Salomon, J.,
and Gourse, R. L.
(1998)
J. Bacteriol
180,
5375-5383 |
44. | Busby, S., and Ebright, R. H. (1999) J. Mol. Biol. 293, 199-213[CrossRef][Medline] [Order article via Infotrieve] |
45. | Stitt, B., and Hinton, D. (1994) in Molecular Biology of Bacteriophage T4 (Karam, J. D., ed) , pp. 142-160, American Society for Microbiology, Washington, D. C. |
46. | Goff, C. G. (1984) Methods Enzymol. 106, 418-429[Medline] [Order article via Infotrieve] |
47. | Marshall, P., Sharma, M., and Hinton, D. M. (1999) J. Mol. Biol. 285, 931-944[CrossRef][Medline] [Order article via Infotrieve] |
48. | Sharma, M., Marshall, P., and Hinton, D. M. (1999) J. Mol. Biol. 290, 905-915[CrossRef][Medline] [Order article via Infotrieve] |
49. | Gerber, J. S., and Hinton, D. M. (1996) J. Bacteriol 178, 6133-6139[Abstract] |