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J Bacteriol, June 1998, p. 3257-3259, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Activation of Bacteriophage Mu mom
Transcription by C Protein Does Not Require Specific Interaction
with the Carboxyl-Terminal Region of the 
or 
70
Subunit of Escherichia coli RNA Polymerase
Weiyong
Sun,1
Stanley
Hattman,1,*
Noboyuki
Fujita,2 and
Akira
Ishihama2
Department of Biology, University of
Rochester, Rochester, New York 14627,1 and
Department of Molecular Genetics, National Institute of
Genetics, Mishima, Shizuoka 411, Japan2
Received 22 December 1997/Accepted 15 April 1998
 |
ABSTRACT |
Late in its growth cycle, transcription of the phage Mu
mom promoter (Pmom) is activated by
the phage gene product, C, a site-specific DNA binding protein. In
vitro transcription analyses showed that this activation does not
require specific contacts between C and the carboxyl-terminal region of
the 
or 
70 subunit of Escherichia coli
RNA polymerase. Unexpectedly, these results are in contrast to those
known for another Mu-encoded transcriptional activator, Mor, which has
a high degree of sequence identity with C and appears to interact with
the carboxyl termini of both and 70.
| |
TEXT |
In addition to the host
Escherichia coli RNA polymerase (RNAP-70)
(22), transcription of the four Mu late operons (including mom) requires the phage-encoded C protein (14, 16, 21,
24, 25, 34, 38, 39). In vitro studies have shown that purified C
alone is capable of activating transcription of the mom
promoter, Pmom (8, 12). It is not
surprising that an accessory protein is required for transcription
because the mom promoter sequence has a poor match to the
consensus E. coli promoter hexamers, TTGACA at 
35 and
TATAAT at 10, and the spacing between them is a suboptimal 19 bp
(13, 15, 32). Although the Mu site has identities of 3 of 6 and 4 of 6 with the consensus 35 and 10 sequences, respectively,
the three matches in the 35 hexamer are not at so-called invariant
positions (28); this has led to the suggestion that the
mom promoter lacks a recognizable 35 sequence (5,
23).
C is a site-specific DNA-binding protein (5, 27) that binds
5' of and adjacent to a poor 35 site in Pmom
and Plys (5, 23). A bipartite
consensus C-recognition site containing partial dyad symmetry,
TTAT_X5,6_ATAACC, has been demonstrated (37).
The location of the C-binding site in Pmom, upstream of and overlapping a poor 35 hexamer, is analogous to that
of several other promoters that are dependent on accessory proteins for
activation, such as the OR2 binding site for 
cI at the
PRM promoter (29) and the binding sites for
AraC (20), OmpR (10), and MalT (30). A
computer-assisted study of 107 E. coli
RNAP-70 promoters revealed that 47 of 48 activatable
promoters have protein binding sites in the region from 65 to +20,
which overlap the RNAP binding site (9), and 30 of those
sites contact the 40 position, as does C. It has been suggested that
the activator proteins for promoters with poor 35 hexamers
functionally substitute for the 35 element in contacting RNAP
(9). Busby and Ebright (7) have noted that many
(but not all) activators that lack an interaction with the
COOH-terminal domain (CTD) of the RNAP subunit bind to sites
overlapping the 35 element. However, it has been shown that the phage
Mu Mor protein, which bears a striking homology to C and binds its
target promoter from 33 to 56, requires contacts with both the CTD and the 70 CTD ( CTD) (2). In this
report, we present evidence that C activation of
Pmom transcription does not involve such an
interaction(s).
(This work was submitted in partial fulfillment of the requirements for
a Ph.D. by W.S.)
Activation of in vitro transcription from the mom
promoter region: lack of interaction between C and the CTD of the RNAP
or 70 subunit.
The sequence of the
mom promoter region is shown in Fig.
1. The C protein binds to
Pmom at a site that partially overlaps the RNAP
site around the 35 region. To investigate possible interaction
between C and RNAP, we analyzed C transcriptional activation of enzyme
reconstituted (11) with a truncated CTD or CTD.
These proteins were purified and characterized previously (11), and their identity was confirmed by sodium dodecyl
sulfate-polyacrylamide gel analysis following shipment to the
laboratory of S.H. (data not shown). For the in vitro studies, we used
a single-round runoff transcription assay (2) and included
DNA containing the modified PRE# promoter of
phage as an internal control; PRE# is a
so-called "extended 10" promoter (6, 19) whose 35
region bears little resemblance to the canonical 35 hexamer. However,
due to interaction of its extended 10 site with region 2.5 of the
RNAP-70 subunit, the PRE#
promoter can be transcribed by RNAP containing a CTD or CTD
truncation (4, 6, 19). In addition, the
PRE# promoter lacks an UP element, an AT-rich
sequence located just upstream of the 35 hexamer in some promoters,
which interacts with the CTD (33). Preliminary control
experiments with the various RNAP forms alone were carried out to
determine the amounts necessary to produce PRE# transcripts at levels comparable with those transcribed by the wild-type (wt) RNAP (data not shown). Thus, the
PRE# promoter served as a reference for
normalizing transcriptional activity at the Pmom
promoter. It should be noted that in the absence of C, wt RNAP produced
some leftward transcripts, but these were no longer observed when C was
present and activated rightward transcription (36a).
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FIG. 1.
Top strand sequence of the mom promoter. The
10 and 35 hexamers are underlined, and the DNase I footprints of C
and RNAP are indicated by horizontal bars; the upstream boundary
protected by RNAP is deduced from the P1 site in the tin7
mutant mom promoter (3,
36).
|
|
The results of in vitro transcription assays with both C and
reconstituted forms of RNAP are shown in Fig.
2A. Note that in
these experiments higher
concentrations of the mutant RNAPs were
necessary to produce
P
RE# transcripts at levels
comparable with
those transcribed by the wt RNAP (Fig.
2A; compare
lanes 3 to 5 with
lanes 6 to 8 and 9 to 12), suggesting lower
efficiency of holoenzyme
assembly with the COOH-terminally truncated
or
70
subunits, or intrinsically lower activity of the mutant enzymes.
The
autoradiographs were computer scanned, and the relative transcriptional
activities of P
mom and
P
RE# are presented
in Fig.
2B (with the
densities of the wt RNAP bands set to 1).
Under the experimental
conditions, RNAP with an
CTD truncation
from amino acid 235 still
retained 62% activity, and 79 to 55%
activities were seen for the
CTD deletions (starting at residues
556 to 529, respectively). These
results demonstrate clearly that
activation of
P
mom transcription does not require specific
interaction between C and the COOH-terminal region of either the
or
the
70 subunit of RNAP.
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FIG. 2.
Gel analysis of single-round in vitro transcription by
reconstituted wt RNAP and mutant RNAPs with COOH-terminally truncated
or 70 subunits. (A) A mix of 5-fmol
PRE# and 3-fmol Pmom
fragments (except for lanes 1 and 2, which contained
PRE# and Pmom alone,
respectively) was incubated with a saturating concentration of C (80 nM) (in a solution containing 25 mM Tris · HCl (pH 7.9), 50 mM
KCl, 5 mM MgCl2, 1 mM dithiothreitol, 3% glycerol, 25 µg
of bovine serum albumin/ml, and 0.05% Nonidet P-40), and then wt or
mutant RNAP was added. Transcription was initiated by the addition of
radioactive [-32P]UTP (40 µM; 8 Ci/mmol), unlabeled
ATP, GTP, and CTP (160 µM each), and heparin (200 µg/ml). After 15 min of incubation at 37°C, the reactions were terminated by addition
of an equal volume (12 µl) of loading dye. Ten microliters of each
sample was electrophoresed on 5% sequencing gels. Amounts of RNAP used
in lanes 1 to 12 were 113, 113, 113, 75, 50, 211, 141, 94, 50, 480, 370, and 800 fmol, respectively. Marker runoff transcripts (lane M)
were generated from the T7 promoter of pBluescript II SK()
(Stratagene) and its derivative (constructed by replacing the
HindIII/SalI fragment with the corresponding
polylinker sequence from pBend2 [18]). The shorter
PRE# transcript (marked with an asterisk) is
presumably due to pausing or premature termination. (B) Relative
transcriptional activities at Pmom and
PRE# for wt and mutant RNAPs. The
autoradiographs were computer scanned, and the densities of the wt RNAP
bands were set to 1. Note that the value for  -235 RNAP was taken
from panel A, lanes 5 and 8.
|
|
Our observations are in contrast to those for the phage Mu
transcriptional activator, Mor, which appears to require interactions
with the COOH termini of both the
and
70 subunits to
activate transcription of the middle promoter
(P
m)
(
2). This was quite surprising
because Mor bears a high degree
of homology to C, and both recognize
imperfect hyphenated AT-rich
inverted-repeat sequences (
12,
26,
37). However, there are
differences in how these proteins
interact with their respective
promoters and with RNAP in vitro. For
instance, C binding to P
mom introduces a DNA
deformation (bending and/or untwisting) and a
new DNase I
hypersensitive site (
31,
36,
37), while Mor
has no such
effects on P
m (
1,
17). Furthermore,
addition of RNAP was shown to introduce an extension of DNase
I
protection 5' to the Mor protection boundary in
P
m (
2), which was attributed to
additional contacts made by the
CTD. In contrast, no such upstream
extension of DNase I protection
was observed with C and RNAP (
12,
36). Thus, whether and how
a transcriptional activator protein
interacts with RNAP may be
influenced by the location of the activator
binding site and the
sequence of the target promoter DNA, as well as
the biochemical
properties and structure of the activator protein
itself. It will
be interesting to examine whether there is any
requirement for
C-RNAP interaction in transcriptional activation of the
other
late Mu promoters, P
lys,
P
P, and P
I.
Although we
cannot rule out the possibility that C interacts with
some other region
of
or
70, or with the
or
' subunit of RNAP,
we currently favor the
notion that C activation of
P
mom transcription is mediated
through a
deformation in DNA structure, which is known to be induced
by C binding
in this region (
31,
36,
37). In this regard,
transcriptional
activation by the Hg(II)-dependent MerR factor
appears to be mediated
by unwinding of
merOP DNA (reviewed in
reference
35), although it is still not clear whether specific
MerR-RNAP interaction is also required. Thus, it remains open
whether
transcriptional activation can be mediated purely through
factor-induced alteration in DNA topology.
| |
ACKNOWLEDGMENTS |
This work was supported by a Public Health Service grant (no.
GM2922, to S.H.) and a Grant-in-Aid from the Ministry of Education, Science, Culture, and Sport of Japan, and by CREST (Core Research for
Evolutional Science and Technology) of Japan (to A.I.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, University of Rochester, Rochester, NY 14627-0211. Phone:
(716) 275-8046. Fax: (716) 275-2070. E-mail:
modDNA{at}uhura.cc.rochester.edu.
| |
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J Bacteriol, June 1998, p. 3257-3259, Vol. 180, No. 12
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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