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Originally published In Press as doi:10.1074/jbc.M107476200 on October 11, 2001
J. Biol. Chem., Vol. 276, Issue 50, 46941-46945, December 14, 2001
DNA Unwinding Mechanism for the Transcriptional Activation of
momP1 Promoter by the Transactivator Protein C of
Bacteriophage Mu*
Shashwati
Basak § and
Valakunja
Nagaraja¶
From the Department of Microbiology and Cell Biology,
Indian Institute of Science, Bangalore 560 012, India and the
¶ Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore 560 064, India
Received for publication, August 5, 2001, and in revised form, October 10, 2001
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ABSTRACT |
Transcription factor-induced conformational
changes in DNA are one of the mechanisms of transcription activation. C
protein of bacteriophage Mu appears to transactivate the
mom gene of the phage by this mode. DNA binding by C to its
site leads to torsional changes that seem to compensate for a weak
momP1 promoter having a suboptimal spacing of 19 bp between
the poor  35 and 10 elements. The C-mediated unwinding could realign
the promoter elements for RNA polymerase recruitment to the reoriented
promoter. In this study, the model has been tested by mutational
analysis of the spacer region of momP1 and by assessing the
strength of the mutant promoters. The response to C-mediated
transactivation was dependent on the spacer length of the promoters.
Mutants with 17-bp spacing between the two promoter elements showed
reduced activity in the presence of the transactivator as compared with
their basal level. A synthetic promoter with near consensus promoter
elements and optimal 17-bp spacing was also tested to evaluate the
model. The results imply a role for C-mediated unwinding in
mom transcription activation.
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INTRODUCTION |
The central step in regulation of gene expression is the
transcription process. In prokaryotes, the transcriptional apparatus consists of the RNA polymerase, the various regulators (activators and
repressors), and the promoter region. The principal regulatory step in
transcription occurs during initiation. The efficiency of transcription
initiation process depends on various factors: 1) the promoter
architecture, 2) the ability of RNAP to bind to the promoter, 3)
isomerization of closed complex to open complex, 4) rapid clearance of
the promoter to allow subsequent binding of more RNAP molecules, and 5)
interaction of regulatory proteins with the promoter DNA and/or RNAP to
facilitate any of the above mentioned steps.
A large number of genes having weak promoter elements are transcribed
only when RNA polymerase is assisted by accessory factors called
activators. Locations of activator binding sites are variable with
respect to their distance from the transcription start site (1).
Activators can either function by facilitating binding of RNA
polymerase at the promoter or at any of the steps subsequent to the
binding, namely, isomerization, promoter clearance, or sometimes during
elongation phase. There are two main mechanisms for
activator-dependent transcription initiation process. In
the first one, a direct communication via protein-protein contact between the activator and one or more subunits of RNAP results in
productive RNAP-promoter interactions (2-4). In the second, activator-induced changes in the DNA structure lead to promoter activation (5). Binding of activators lead to DNA conformational changes such as DNA bending, looping, and unwinding (5, 6). Restructuring of the DNA allows favorable alignment of the
cis-elements for productive RNAP-promoter and -activator
interactions leading to transcription activation.
The extent of homology of the 10 and 35 elements to the consensus
sequence and the length of the spacer region in between them determine
the strength of a promoter. Accordingly RNA polymerase alone or in
conjunction with both cis-elements and
trans-acting factor(s) initiates the process of
transcription. The bacteriophage Mu mom operon, which is
responsible for a unique DNA modification function (7), contains two
promoters, momP1 and momP2 (see Fig.
1A and Refs. 8 and 9). The promoter of the mom
gene, momP1, is a typical example of a weak promoter with a
poor 35 hexamer and suboptimal spacing of 19 bp between the 10 and
35 elements (see Fig. 1A). RNA polymerase is not able to
bind to momP1 on its own (8); instead RNAP binds to
momP2 (see Fig. 1A) directing leftward
transcription (8, 9). Optimum transcription initiation at
momP1 requires the binding of the transactivator protein C
to its recognition sequence, 28 to 57 (see Fig. 1A and
Refs. 10 and 11) as a dimer (12).
Site-specific interaction of C protein results in high degree of
distortion and localized unwinding of DNA (13-15). We had proposed a
model for the C protein-mediated transcription activation of the
mom gene (15). An additional twist of 34° caused by 19-bp spacing of the momP1 promoter would result in positioning of
the 10 and 35 hexamers out of phase with respect to each other. The
C protein-mediated torsional changes (unwinding by ~30°) could reorient the promoter elements to a favorable conformation for RNAP
recruitment to momP1. The above hypothesis could be tested by analyzing promoter spacing mutants. In an optimally spaced (17 bp)
promoter one would expect constitutive expression. However, as a result
of activator binding and unwinding, the optimally spaced promoter
should show less activity, because the promoter elements will be
reoriented in an unfavorable position for RNAP recognition and
occupancy. In this study, a series of spacing mutants of
momP1 have been used to verify the unwinding model for
transcription activation.
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EXPERIMENTAL PROCEDURES |
Strains, Plasmids, Primers, Enzymes, and Chemicals--
Plasmids
pVN184, pLW4, and pUW4 have been described earlier (8, 16).
Escherichia coli DH10B was used for all of the cloning experiments. The details about the primers used are available upon
request. Restriction and modifying enzymes were purchased from
Stratagene and Roche Molecular Biochemicals. E. coli DNA polymerase (Klenow fragment) was from New England Biolabs. Superscript reverse transcriptase was purchased from Life Technologies, Inc. Chemicals and other reagents were purchased from Life Technologies, Inc. and Sigma. [ -32P]ATP (6000 Ci/mmol) was purchased
from PerkinElmer Life Sciences. Routine DNA manipulations were carried
out as described (17).
Construction of Spacer Mutants of momP1--
Plasmid pUW4 (16)
was used as template for the PCR-based mutagenesis methods. The mutants
p18, p17, p16, and p15 (see Fig. 1B) were generated by using
the Stratagene QuickChangeTM site-directed mutagenesis
method involving a pair of mutagenic oligonucleotides and
PfuI DNA polymerase. A Promega Gene Editor site-directed
mutagenesis kit was used to generate the mutants p20, p18A, and p17A.
Mutants pWT-P2, p17A-P2, p17A.T2G, and p17P-1 were generated by using a
modified mega primer method (18) as described earlier (19). All of the
mutants generated in the pUW4 background were subcloned into pLW4 using
EcoRI and BamHI restriction enzymes to generate
the promoter mutants as lacZ fusions. Sanger's dideoxy
method of sequencing (17) was carried out to confirm the mutants.
In Vivo Promoter Strength Analysis--
Promoter strength was
analyzed by fusing the various mutant promoters of momP1
with LacZ gene and assaying for the reporter gene activity. Isolated
colonies harboring either a promoter mutant plasmid alone or with
plasmid pVN184 (C protein-producing plasmid) were inoculated into LB
broth containing 100 µg/ml of ampicillin (for mutant promoter
plasmids alone) or ampicillin and 25 µg/ml chloramphenicol (both
plasmids present). An overnight grown culture was used as preinoculum.
The 3.0-ml cultures (in duplicate) were grown till they reached an
A600 of 0.3-0.7. The samples were
chilled on ice. The cells were treated with SDS-CHCl3, and
 -galactosidase activity was determined as described earlier (20).
The values in Table I are the averages from three separate experiments, and the assays were done in duplicate for each culture. The variation was from 10 to 25% about the mean value.
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RESULTS |
In Vivo Activity of momP1 Spacer Mutants--
The spacing between
the 10 and 35 elements of momP1 was altered by either an
insertion or a deletion of base(s) using appropriately designed
mutagenic oligonucleotides (as described under "Experimental Procedures"; Fig. 1B). Thus,
promoters with 20-, 18-, 17-, 16-, and 15-bp spacing were generated
(Fig. 1B). Two different 18- and 17-bp spacing mutants were
created to account for the effect of base-specific deletion(s) on
promoter activity. Promoter strength was measured in terms of
-galactosidase activity of the wild type mom and spacer
mutant promoters in the absence and presence of C protein (Table
I). The response to C-mediated activation was different in the various mutants depending on the spacer length. Change in spacer length by 1 bp (i.e. 20 and 18 bp) leads to
a decrease in the transactivated levels by 3-5-fold as compared with
the wild type promoter activity in the presence of C protein. In two
different 17 bp spacing mutants, 3-10-fold decrease in -galactosidase activity was observed in the presence of C protein as
compared with their basal levels (Table I). These data support the
unwinding model. C protein-mediated unwinding in a 17-bp spacer promoter mutant would result in reorientation of the promoter elements
in an unfavorable position. This would result in less activity of the
promoter as compared with its constitutive level. However, the 16- and
15-bp spacer length mutants showed an increase in C-mediated
transactivation values by 41- and 4-fold respectively (Table I). This
is due to the generation of a new 10 element (GATAGT) as a
consequence of deletions made in the spacer region of the promoter.
Formation of the new 10 hexamer was confirmed by mapping the +1 start
site, which was found to be shifted by two bases downstream in the case
of the 16-bp spacing mutant as compared with that of wild type
momP1 transcript (not shown). Thus, the actual spacing in
the case of the 16- and 15-bp spacer mutants are 19 and 18 bp,
respectively. The 15-bp spacer mutant (actual spacing 18 bp) shows less
activity in the presence of C when compared with the transactivated
level of the 16-bp spacing mutant (actual spacing 19 bp). These mutants
essentially follow a similar pattern as that of the wild type and 18-bp
spacer deletion mutants (Table I). In all of the cases, however, the
change in the spacer length by either an insertion of a base or by a
deletion of base(s) did not alter their constitutive activities. The
reason for this has been discussed in the section below.
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Fig. 1.
A, sequence of the Mu mom
regulatory region. The 10 and 35 elements of momP1
promoter and the 10 box of momP2 promoter are indicated.
The momP2 promoter does not have a recognizable 35
element. The C binding site is shown as an open rectangle.
Transcription start sites of both the promoters are indicated with
arrows. The stretch of six T residues is shown with a
dotted line. The RNA polymerase binding regions at both the
promoters are marked with thick lines. B, mutants
of momP1 promoter. Insertion and deletion mutations within
the spacer region of momP1 promoter are indicated. The 10
and 35 hexamers of momP1 are shown with open
boxes.
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Table I
Production of -galactosidase activity in E. coli DH10B cells
containing a pLW4-momP1 promoter-lacZ fusion derivative with or without
compatible plasmid pVN184
See "Experimental Procedures" for growth of cells and enzyme
assays. Plasmid pVN184 produces C protein constitutively at a low
level. E. coli DH10B cells alone and harboring pVN184 do not
show any enzyme activity.
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Optimally Spaced 10 and 35 Elements Are Not Sufficient to
Increase the Basal Activity of the momP1 Promoter--
Absence of high
level of constitutive activity in the optimally spaced momP1
promoter could be attributed to three possibilities: 1) RNA polymerase
preferentially binds to momP2 instead of momP1 in
the absence of C protein (8, 9) even when the promoter elements are
optimally spaced; 2) the putative, weak 35 element of
momP1 (3 of 6 match at the least consensus positions) may
not be functional; and 3) the negative element present in the form of
T6 run in the spacer region of momP1 prevents
transcription initiation at the promoter in the absence of the
transactivator (19).
Effect of Divergent Promoter, momP2, on 17-bp Spacer Deletion
Mutant of momP1--
To test the effect of the divergent promoter
(momP2) on the transcription of the 17-bp spacer mutant of
momP1, the 10 element of momP2 was disrupted by
site-directed mutagenesis in the case of both the wild type and
17A spacer deletion mutant promoters (Fig.
2A). Disruption of
momP2 was confirmed by the lack of transcript formation from
the momP2 promoter (19). The -galactosidase levels of the
wild type and the 17A spacer deletion mutant were assayed in the
background of momP2 disruption and compared with the basal
level of promoter activity obtained when momP2 is intact (Fig. 2B). There was no increase in the basal activity of
both the promoters in the momP2 10 hexamer mutated
background. These results demonstrate that the nonfunctional
momP2 promoter does not enhance the constitutive level of
momP1, and hence, the leftward promoter has a negligible
role to play in the activity of momP1 promoter.
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Fig. 2.
Promoter activity of
momP1. A, momP2 10
element mutation effect. The sequence of mom regulatory
region is depicted with the mutation in the 10 box of
momP2 in WT and 17A spacer deletion mutant background.
B, promoter strength of momP1 mutants with
disrupted 10 hexamer of momP2. C, effect of
mutations in the 35 element of momP1. The sequence of the
mom regulatory region is shown indicating the position of
mutations in the 35 box of momP1. D, promoter
strength of momP1 with a perfect 35 element compared with
the wild type momP1 35 box sequence. The values are the
averages of at least three different experiments.
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Effect of Mutations in the 35 Element of momP1--
To ascertain
the presence of a functional 35 element with a spacing of 19 bp at
momP1 promoter, the 35 hexamer (ACCACA) was mutated to
obtain a perfect 35 box, TTGACA (Fig. 2C). Fig. 2D shows the result of the promoter strength analysis.
Constitutive activity of the momP1 promoter with a perfect
35 element was increased by ~18-fold when compared with the wild
type promoter activity. The transactivated level of this promoter in
the presence of C protein could not be assessed as the C binding site
gets disrupted because of the mutations made in the 35 box. Hence, in
the 17-bp mutant, one of the possible reasons for the low constitutive activity could be attributed to having a weak 35 element.
Effect of Disruption of T6 Run on the Basal Level
Promoter Activity of 17-bp Spacer Deletion Mutant--
The presence of
a run of six T nucleotides next to the 10 element (Fig.
1A) imparts an unfavorable distortion to the
momP1 promoter (19). When the T stretch was disrupted by
substitution with other bases, some of the mutants showed an increase
in the basal level activity by 4-10-fold as compared with the wild
type promoter. To assess the effect of the T stretch on the basal
activity of the 17-bp spacer deletion mutant, 16T was substituted
with G to disrupt the T6 run in the 17A mutant (Fig.
3A). As a control, T2G mutant
having 19-bp spacing and T to G substitution at the 16 position (19)
was used for the promoter strength analysis. There was an increase in
the basal activity of the 17-bp spacer mutant with a disrupted T
stretch (17A.T2G) by 10-fold as compared with the 17-bp promoter mutant
with an intact T6 run (i.e. 17A) (Fig.
3B and Table I). In the case of T2G mutant, there was an increase in the activity by 8-fold wherein the spacing between the
promoter elements is 19 bp (Fig. 3B and Table I).
Removal of the negative element increases the constitutive activity of the promoter irrespective of the spacer length between the promoter elements. Hence, the presence of the T6 stretch within the
spacer region of the 17-bp spacer mutant is the major factor
responsible for its low level promoter strength.
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Fig. 3.
Effect of disruption of the T6
run on the activity of 17A spacer deletion mutant. A,
sequence of momP1 regulatory region with the mutation in the
T6 run indicated. B, -galactosidase levels of
the momP1 mutants in the absence and presence of C
protein.
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However, the response of the promoters having different spacer length
(19 and 17 bp) to transactivation by C produced contrasting results.
The T2G mutant showed a 12-fold increase in the transactivated level as
compared with its basal activity (Fig. 3B). In contrast to
this, the 17A.T2G mutant promoter showed 2-fold less activity than its
basal value in the presence of C protein (Fig. 3B),
essentially supporting the proposed role for C protein.
Effect of C Protein on a Promoter Having a Strong 35 Element and
Optimal Spacing--
A test for the role of C protein-mediated
unwinding in transactivation would be its ability to influence
transcription by the same mechanism on synthetic promoters.
Transcription from a promoter with optimally spaced (17 bp) elements
should be alleviated in the presence of C protein bound to its
recognition sequence positioned next to the 35 element. The design
and construction of such a template is presented in Fig.
4A. Attempts to clone a
synthetic promoter with a perfect 35 element (TTGACA) and 17-bp optimal spacing in pLW4 background were not successful. Different types
of promoter down-mutations were encountered consistently while cloning
such a promoter. Thus, the TTGACA sequence was replaced with TCGACA and
17-bp spacing in between the promoter elements with the C binding site
located next to the 35 element (Fig. 4A). As expected, the
promoter with better consensus and 17-bp spacing exhibits high
constitutive activity (Fig. 4B). The 2-fold decrease of the
promoter activity in the presence of C protein is a strong support for
the C-mediated DNA unwinding mechanism.
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Fig. 4.
A, schematic representation of the
design of a synthetic promoter. The wild type 35 hexamer (ACCACA)
is replaced with TCGACA, and the C protein binding site is placed next
to the 35 element using appropriate oligonucleotides as described
under "Experimental Procedures." The spacing between the promoter
elements is 17 bp. B, strength of the synthetic promoter
(17P-1) and the wild type momP1 promoter (WT) in
the absence and presence of C protein.
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DISCUSSION |
In this paper, we have addressed the mechanism of C-mediated
transactivation of the mom gene of bacteriophage Mu (15). C binding to its site leads to DNA unwinding possibly leading to reorientation of promoter elements, thus allowing RNA polymerase to
bind to an erstwhile inaccessible promoter. Evaluation of this proposal
was carried out by analyzing the effect of promoter element spacing of
momP1 on the constitutive as well as the transactivated levels of promoter activity. Change in the length of the spacer region
between the 10 and 35 elements affected the activity of the mutant
promoters in the presence of C protein. With an optimal spacing (17 bp), the promoter shows less activity in the presence of C protein as
compared with its basal level (Table I and Figs. 3B and
4B). These results lead us to conclude that C-mediated
momP1 transactivation requires a 19-bp spacing between the promoter elements.
The local DNA structure seems to influence the transcription process by
providing proper conformation for the recognition and binding of RNA
polymerase to promoter sequences. The spacer region of a promoter is
believed to play an important role in the fine tuning of promoter
activity both by virtue of its length and sequence (21-25). The role
of the spacer DNA is postulated to position the 10 and 35 elements
in a favorable orientation for RNA polymerase recognition without
having any base-specific contacts with RNA polymerase. The spacer
region of momP1 has significant functions in the regulation
of transcription initiation of mom gene. A lack of increase
in the basal level activity of the optimally spaced promoter constructs
implies the importance of the T6 run as well as suboptimal
spacing along with a weak 35 box in ensuring repression of
momP1 to prevent leaky expression of mom product. Complete silencing of mom is necessary until the late lytic
phase of the phage because premature mom modification would
be cytotoxic to the host cell. The role of C is, thus, to counter the
negative regulation mediated by cis-elements in the promoter region.
A suboptimal spacing of 19 (and sometimes 20) bp between the 10 and
35 elements is found in some E. coli promoters. Many promoters that are dependent on transactivators responsive to metal
ions have suboptimal spacing between their promoter elements. MerR is
one such well characterized transcription activator that unwinds DNA in
a symmetric fashion in response to Hg(II) leading to RNAP recruitment
to merT promoter (26, 27). Proteins belonging to the MerR
family like SoxR (28), ZntR (29), and CueR (30) are other examples that
transactivate promoters having suboptimal spacing between their 10
and 35 elements. Some of these activator proteins like SoxR (28) and
ZntR (29) have been shown to act via a DNA distortion/unwinding
mechanism like that of MerR to activate transcription of their
respective promoters. Based on the similarity in the pattern of
transcription activation by C protein at momP1, it can also
be grouped along with the MerR class of activators.
Although, mechanistically C protein appears to be similar
to the MerR family of regulators, there are a number of differences in
the interaction of C protein at its site as compared with the MerR
class of proteins. Members of the MerR family bind to sites within the
spacer region of the promoter between the 10 and 35 elements (Fig.
5). The binding site of C is upstream of
the 35 element, with a partial overlap with the sequence (Figs.
1A and 5). This difference in the position of the
activator-binding site probably accounts for the distinct features of
C-mediated transactivation that is different from that of MerR and
related transcription factors. In the presence of the metal inducer,
all three proteins (MerR, SoxR, and ZntR) produce hypersensitive bands
at the center of the palindromic binding site upon 5-phenyl-OP-Cu
footprinting (29, 31, 32). (OP)2Cu footprinting with both
supercoiled and linear templates harboring the C binding site produced
hypersensitive bands only in the 3' half-site of the C binding site
(13, 15). Also, dimethyl sulfate protection and interference
footprinting analysis reveal asymmetric interaction of C at the two
half-sites of its recognition sequence (13, 15). Such an asymmetric
interaction and unwinding of DNA by C protein seems to be necessary
because of its location with respect to the 10 and 35 promoter
elements in contrast to the MerR mode of interaction (Fig. 5). Upon
recruitment of RNA polymerase, in the case of merT promoter,
both MerR and RNAP would be bound facing each other on opposite faces
of DNA, whereas at momP1, C and RNAP would perhaps bind on
the same face of DNA alongside each other. In this context, MerR is
shown to contact three different subunits of RNAP:  , , and
 70, in cross linking experiments (33). On the other
hand, one would expect C to interact with or 70
subunits based on the location of its binding site. However, previous
studies indicate that C does not interact with either one of them (34),
and its interaction with other subunits, if any, is yet to be
addressed.
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Fig. 5.
A, C protein binds to its site
overlapping the 35 element. Asymmetric unwinding by C protein
realigns the unfavorably positioned promoter elements in proper
orientation for recognition and binding by RNA polymerase.
B, binding of MerR within the spacer region of its promoter
results in symmetric unwinding and repositioning of its promoter
elements for RNA polymerase occupancy.
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In the case of MerR, the protein acts as a repressor of the
merT promoter in the absence of the metal ion Hg(II),
although MerR is still able to bind to its site without affecting the
binding of RNA polymerase (31). On the other hand, the SoxR protein does not repress the soxS promoter under similar conditions.
C protein in the presence of Mg(II) undergoes mandatory conformational changes for binding to its site specifically (35) to activate the
mom gene. However, in the absence of Mg(II), it does not
bind DNA, unlike MerR. Instead, the momP1 promoter has a DNA
negative element within the spacer region near the 10 element that
keeps the promoter in the inactive state in the absence of C protein (19).
To summarize, in the absence of the transactivator protein, C, RNA
polymerase engages in leftward transcription from momP2 promoter. Inability of RNAP to bind to momP1 is due to the
presence of a weak 35 element, suboptimal spacing, and a negative
element within the spacer region of momP1. Binding of C in
the presence of Mg(II) to its specific site results in unwinding of DNA
at the 3' half-site of its binding region. This alteration in the DNA
structure appears to compensate for the presence of two extra bases
within the spacer region and also probably for the intrinsic DNA
distortion. These changes in the promoter lead to RNAP recruitment at
momP1. From undetectable expression levels, mom
gene is turned on completely by the binding of C. Thus, C acts as a
decisive transactivation switch operating at the late phase of the
phage lytic cycle. The Mu mom promoter has evolved in such a
way that the architecture of the promoter assures a tight regulation of the gene by utilizing both phage and host-encoded proteins (36) in
addition to the DNA structure (19). Such a tight and intricate regulation of the mom gene is essential for the survival
of both the phage and the bacterial cell.
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ACKNOWLEDGEMENTS |
We thank Jibi Jacob for technical assistance
and Veena Rao for critical reading of the manuscript.
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FOOTNOTES |
*
This work was supported by a grant from the Department of
Science and Technology, Government of India (to V. N.).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.
§
Recipient of Jawaharlal Nehru Center for Advanced Scientific
Research, Bangalore.
To whom correspondence should be addressed: Dept. of
Microbiology and Cell Biology, Indian Inst. of Science, Bangalore 560 012, India. Tel.: 91-80-360-0668; Fax: 91-80-360-2697; E-mail: vraj@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M107476200
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