Download Two DNA sites for MelR in the same orientation are sufficient for

RESEARCH LETTER
Two DNA sites for MelR in the same orientation are sufficient
for optimal MelR-dependent repression at the Escherichia coli
melR promoter
Mohamed S. Elrobh1,2, Christine L. Webster3, Shivanthi Samarasinghe3, Danielle Durose3 &
Stephen J.W. Busby3
1
Biochemistry Department, College of Science, King Saud University, Riyadh, Saudi Arabia; 2Biochemistry Department, Faculty of Science,
Ain Shams University, Cairo, Egypt; and 3School of Biosciences, University of Birmingham, Birmingham, UK
Correspondence: Stephen Busby, School of
Biosciences, University of Birmingham,
Edgbaston, Birmingham B15 2TT, UK.
Tel.: 44 121 414 5439; fax: 44 121
414 5925; e-mail: [email protected]
Received 31 July 2012; revised 3 October
2012; accepted 4 October 2012. Final
version published online 8 November 2012.
DOI: 10.1111/1574-6968.12027
Abstract
The Escherichia coli melR gene encodes the MelR transcription factor that controls melibiose utilization. Expression of melR is autoregulated by MelR, which
represses the melR promoter by binding to a target that overlaps the transcript
start. Here, we show that MelR-dependent repression of the melR promoter
can be enhanced by the presence of a second single DNA site for MelR located
up to 250 base pairs upstream. Parallels with AraC-dependent repression at the
araC–araBAD regulatory region and the possibility of the MelR-dependent
repression loop formation are discussed. The results show that MelR bound at
two distal loci can cooperate together in transcriptional repression.
MICROBIOLOGY LETTERS
Editor: Olga Ozoline
Keywords
Escherichia coli; melibiose operon regulatory
region; promoters; DNA sites for MelR;
repression; loop formation.
Introduction
The activity of many bacterial promoters is controlled by
transcription repressors, and many cases have now been
described where efficient repression requires interaction
between repressors bound at two separated DNA targets,
resulting in looping of the intervening DNA (Browning &
Busby, 2004). One of the first cases to be described was
repression by the Escherichia coli AraC protein at the
araC-araBAD intergenic regulatory region, which requires
AraC binding to two target sites, I1 and O2, separated by
210 base pairs (reviewed by Schleif, 2010).
In previous work, we have studied the interactions of
MelR at the E. coli melibiose operon regulatory region
(Wade et al., 2000, 2001). MelR is a member of the AraC
family of transcription factors and is essential for melibiose-dependent triggering of the melAB operon that
encodes products needed for melibiose catabolism and
transport. The melR gene is located upstream of the
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
melAB operon, and the melR and melAB promoters
are divergent, with the transcript start sites separated by
256 base pairs (Webster et al., 1987). We showed that
transcription activation at the melAB promoter requires
binding of MelR to four target sites (denoted 1′, 1, 2 and
2′) and binding of the global regulator, cyclic AMP receptor protein (CRP) to a target site located between MelR
sites 1 and 2 (Fig. 1a). Transcription initiation at the
melR promoter is dependent on activation by CRP and is
repressed by MelR binding to a single target site (denoted
R) overlapping the melR transcript start. Wade et al.
(2000) reported that efficient MelR-dependent repression
of the melR promoter requires upstream sequences that
covered the melAB promoter and that the most important
element in repression is MelR binding at target site 2.
Further detailed analysis by Samarasinghe et al. (2008)
showed that MelR bound at sites 1 and 1′ plays a role
in repression, and images from atomic force microscopy
suggested that repression is due to a nucleoprotein
FEMS Microbiol Lett 338 (2013) 62–67
Optimal repression by E. coli MelR.
63
melR
promoter
(a)
melAB
2’ 2
1 1’
R
melAB
promoter
–174.5
2
(b)
1
melR
+2.5
R
1’
TB22
TB23
TB31
TB28
TB33
–174.5
+2.5
% Activity
(c) 150
–MelR
+MelR
100
50
0
TB22
TB23
TB31
TB28
TB33
Fig. 1. The Escherichia coli melibiose operon regulatory region and
MelR-dependent repression of the melR promoter. (a) Schematic
diagram of the intergenic region between the divergent melAB and
melR transcription units. The bent arrows indicate the transcription
start sites for the melAB and melR promoters, and triangles denote the
corresponding -10 hexamer elements. The locations of the different
18-bp DNA sites for MelR (sites 2′, 2, 1, 1′ and R) are indicated by
shaded rectangles, and the DNA sites for CRP are denoted by pairs of
ovals. (b) Diagram of the starting TB22 EcoRI–HindIII melR promoter
fragment carrying the segment of the melibiose operon regulatory
region indicated by the dotted lines joining panels A and B, drawn the
same symbols as in panel A. The figure also illustrates the TB23, TB31,
TB28 and TB33 derivatives, with substituted sequences denoted by the
thickened black line. The orientation of each of the DNA sites for MelR
is denoted by a horizontal arrow, and the locations of centre of some
of these sites are indicated. (c) The figure illustrates measured levels of
b-galactosidase in E. coli WAM1321 cells carrying melR promoter::lac
fusions, encoded by pRW50 with the indicated promoter fragments.
b-Galactosidase expression was measured in cells containing plasmid
pJW15 that encodes MelR (+MelR: black filled bars), or the control
pJW15DmelR plasmid with no melR insert ( MelR: open bars) as
outlined in Materials and methods. For each promoter, the activity
+MelR is expressed as a% of the activity MelR. The results are
averages from at least three independent determinations.
complex consisting of four MelR subunits and ~170 base
pairs of DNA between MelR-binding target site 2 and
target site R.
FEMS Microbiol Lett 338 (2013) 62–67
Most members of the AraC family of transcription regulators function as homodimers of two subunits with the
N-terminal domain of each subunit involved in ligand
binding and dimerization, and the C-terminal domain
responsible for DNA binding (Gallegos et al., 1997).
C-terminal domains of AraC family members are highly
conserved, carry two helix-turn-helix motifs and bind to
asymmetric ~18 base pair target operator sequences. As it
is well established that effective transcriptional repression
can result from the two subunits of a single AraC dimer
binding to two separated target sites (Schleif, 2010), and
as MelR has been shown to dimerise (Bourgerie et al.,
1997; Kahramanoglou et al., 2006), we revisited the E. coli
melibiose operon regulatory region to investigate whether
two DNA sites for MelR could be manipulated to produce efficient MelR-dependent repression of the melR
promoter.
Materials and methods
In this work, we exploited the low-copy-number lac
expression vector plasmid, pRW50, encoding resistance to
tetracycline (Lodge et al., 1992). The starting points of
the work were pRW50 derivatives carrying the TB22 and
TB23 EcoRI-HindIII fragments (Fig. 1b) containing the
E. coli melR promoter, as described by Samarasinghe
et al. (2008). These recombinant pRW50 derivatives each
carry a melR promoter::lacZ fusion, and they were propagated in the WAM1321 E. coli K-12 Dlac Dmel strain to
measure melR promoter activity. Cells were grown in
minimal medium with fructose, as a carbon source, and
35 lg mL 1 tetracycline, as in the study by Samarasinghe
et al. (2008), and the Miller (1972) method was used to
quantify b-galactosidase expression. For the different
melR promoter fusions studied here in our conditions in
the absence of MelR, b-galactosidase activity levels range
from 360 to 400 standard Miller units. To quantify
repression by MelR, cells also carried pJW15, encoding
melR or empty vector, pJW15DmelR, and 80 lg mL 1
ampicillin was included in the media, as described by
Kahramanoglou et al. (2006). In experiments to measure
effects due to MalI, cells also carried pACYC–malI,
encoding malI or empty vector, pACYC-DHN (Lloyd
et al., 2010), and 10 lg mL 1 chloramphenicol was
included in the media.
Derivatives of the TB22 and TB23 EcoRI-HindIII fragments, illustrated in Figs 1–4, were constructed by standard recombinant DNA technology using synthetic oligos
purchased from Alta Bioscience (http://www.altabioscience.com/) and cloned into pRW50. The complete
annotated base sequence of each fragment is listed in the
Data S1 (Supporting information), and the DNA
sequences were checked by the functional genomics facility
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Published by Blackwell Publishing Ltd. All rights reserved
64
of the University of Birmingham College of Life and Environmental Sciences (http://www.genomics.bham.ac.uk/).
Results and discussion
Optimal MelR-dependent repression due to
two DNA sites for MelR in the same
orientation
To investigate MelR-dependent repression at the melR
promoter, we exploited different melR promoter::lac
fusions carried by derivatives of the pRW50 lowcopy-number lac expression plasmid, and b-galactosidase
expression was measured in the WAM1321 E. coli K-12
Dlac Dmel host strain, containing either plasmid pJW15,
encoding melR or empty vector. The starting experiment
compared MelR-dependent repression of the melR promoter carried on the TB22 and the TB23 fragments, illustrated in Fig. 1b. The 251 base pair TB22 EcoRI-HindIII
fragment carries DNA sequence from 192 base pairs
upstream of the melR promoter transcript start (position
192) to 59 base pairs downstream (+59) and includes
MelR target site 2, whilst in the 227 base pair TB23 fragment, MelR target site 2 is deleted. Results illustrated in
Fig. 1c show that, as expected, the deletion of site 2 in
the TB23 fragment causes a clear reduction in MelRdependent repression of the melR promoter and confirms
previous observations (Wade et al., 2000).
Previously, we identified the DNA target site for MelR
subunits as an 18 base pair asymmetric sequence (Webster et al., 1987; Wade et al., 2001). By convention, we
denote the location of each site by its centre with respect
to the target promoter. Hence, at the melR promoter,
MelR-binding site R is located at position +2.5 (i.e.
between base pairs 2 and 3 downstream from the melR
promoter transcript start) and MelR-binding site 2 is
located at position 174.5 (i.e. between base pairs 174
and 175 upstream from the melR promoter transcript
start). To investigate whether the binding of two MelR
subunits could be sufficient to repress the melR promoter
efficiently, we constructed the TB31, TB28 and TB33 fragments, illustrated in Fig. 1b. TB31 carries the core melR
promoter sequences exactly as in TB22 and TB23, but
DNA sequence upstream of position 80 is replaced by
unrelated sequence. TB28 and TB33 are derivatives of
TB31 carrying a single consensus 18 base pair site for
MelR at position 174.5. In the TB28 fragment, this site
has the same orientation as site 2 in the starting TB22
fragment, whilst, in TB33, this site has the opposite orientation, which is the same as for site R. Results illustrated in Fig. 1c show that MelR-dependent repression of
the melR promoter in the TB31 and TB28 fragments is
weak, but is increased to ~90% with the TB33 fragment.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M.S. Elrobh et al.
The weak repression of the melR promoter carried on
the TB31 fragment must be due to MelR binding to the
target site R alone, and this result is consistent with the
study by Wade et al. (2000). The ~90% repression found
with the TB33 fragment must be due to MelR binding to
the targets at both positions 174.5 and +2.5 and interaction between MelR bound at the two loci. Strikingly,
repression is greatly reduced with the TB28 fragment
(Fig. 1b and c), and this was expected from our previous
work in which we replaced MelR target sites 1 and 1′ and
the adjacent DNA site for CRP (Samarasinghe et al.,
2008). Hence, as for AraC-dependent repression at the
araC-araBAD intergenic region, efficient repression with
just two bound regulator molecules depends on both target sequences being in the same orientation (Carra &
Schleif, 1993).
MelR-dependent repression at the TB33 melR
promoter is insensitive to spacing but reduced
by a decoy DNA site for MelR
The centre-to-centre distance between the two DNA sites
for MelR in the TB33 fragment is 176 base pairs. To
investigate the relation between spacing and repression,
we constructed a series of derivative fragments with the
upstream MelR target at different locations, ranging
from position 254.5 to position – 83.5. This is illustrated in Fig. 2, which also lists the percentage MelRdependent repression for each case. The data show that
repression is largely unaffected as the upstream DNA site
for MelR is moved through ~170 base pairs, including
translocation by five base pairs to the opposite face of
the DNA helix (compare repression with TB33, TB332
and TB333).
A simple explanation for our observations is that
repression of the melR promoter in the TB33 fragment
and its derivatives is due to a bridging interaction
between MelR bound at the upstream and downstream
DNA sites and subsequent loop formation, and this interaction must be sufficiently flexible to accommodate different distances and different face of the DNA helix
juxtapositions between the sites. We suppose that the lack
of efficient repression with the TB23 fragment (Fig. 1c)
must be due to interactions between MelR bound at site
1 and site 1′ that preclude interaction with site R
(Fig. 1b). To investigate this, we constructed the TB33P
and TB33R derivatives illustrated in Fig. 3a. These fragments are derivatives of TB33 that contain a supplementary upstream DNA site for MelR organised in either the
same orientation (TB33P) or opposite orientation
(TB33R). Results illustrated in Fig. 3b show that the presence of the supplementary DNA site for MelR significantly reduces MelR-dependent repression of the melR
FEMS Microbiol Lett 338 (2013) 62–67
Optimal repression by E. coli MelR.
65
%
MelR-dependent
Repression
TB344 (–254.5)
89
TB342 (–214.5)
87
TB341 (–194.5)
84
TB33 (–174.5)
90
TB332 (–169.5)
92
TB333 (–164.5)
94
TB334 (–136.5)
88
TB331 (–123.5)
95
TB335 (–116.5)
90
TB336 (–103.5)
82
TB337 (–83.5)
92
TB31
28
+2.5
TB33
–194.5
TB33P
–204.5
Fig. 2. MelR-dependent repression of the melR promoter and
upstream-bound MelR. The figure illustrates a series of EcoRI-HindIII
melR promoter fragments derived from TB33, with the upstream DNA
site for MelR at different locations as listed in brackets. Conventions
for the different symbols are as in Fig. 1. The numbers in the column
on the right side of the figure denote the% repression of melR
promoter activity by MelR, measured as outlined in the legend to
Fig. 1.
promoter, presumably because the supplementary site acts
as a decoy for MelR–MelR interactions.
Effects of MalI binding between MelR targets
on MelR-dependent repression
The flexibility in the spacing of the two DNA sites for
MelR observed in the experiment illustrated in Fig. 2 suggested that it would be interesting to insert an intervening
site for another DNA-binding protein. In recent work,
Lloyd et al. (2010) identified the DNA site for the E. coli
MalI repressor (that is a member of the LacI family) as a
symmetric 16 base pair sequence element. Figure 4a illustrates an experiment where one or two of these elements
were inserted between the two DNA sites for MelR in the
TB334 fragment. Results in Fig. 4b show that in the
absence of a plasmid encoding MalI, as expected, these
insertions have but small effects on MelR-dependent
repression of the melR promoter. However, with plasmid
pACYC-malI, which encodes MalI, there is a clear small
significant relief of repression with the TB334I-1 and
TB334I-2 fragments carrying one or two MalI operator
elements, but no relief with the control TB31, TB33 or
TB334 fragments.
FEMS Microbiol Lett 338 (2013) 62–67
–174.5
(a)
TB33R
TB31
(b) 120
–MelR
+MelR
% Activity
100
80
60
40
20
0
TB33
TB33P
TB33R
TB31
Fig. 3. Decoy MelR affects MelR-dependent repression of the melR
promoter. (a) Schematic diagram of EcoRI-HindIII melR promoter
fragments derived from TB33 with an upstream decoy DNA site for
MelR. Conventions for the different symbols are as in Fig. 1. (b) The
figure illustrates b-galactosidase expression in Escherichia coli
WAM1321 cells carrying melR promoter::lac fusions, encoded by
pRW50 with the indicated promoter fragments. Expression was
measured in cells containing plasmid pJW15 (+MelR: black filled bars)
or the control pJW15DmelR plasmid (-MelR: open bars) as outlined in
Materials and methods. For each promoter, the activity +MelR is
expressed as a% of the activity MelR, and each result is the average
from at least three independent determinations.
Conclusions
The expression of many transcription repressors is autoregulated by repression (Browning & Busby, 2004). Kahramanoglou et al. (2006) proposed a two-state model for
MelR in which, in the absence of its ligand, melibiose,
MelR acts as an autorepressor of its own production by
repressing the melR promoter. Samarasinghe et al. (2008)
showed that this repression was due to the formation of a
nucleoprotein complex involving four MelR subunits.
Here, we report that it is possible to construct simpler
derivatives of the melR promoter where only two MelR
targets are needed for efficient repression (Fig. 1), and
there are clear parallels between this and AraC-dependent
repression at the araC–araBAD intergenic region, where
repression is dependent on interaction between two AraC
subunits bound to targets separated by 210 base pairs
(Schleif, 2010). An explanation for the observed repression with the TB33 fragment is that MelR subunits bound
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Published by Blackwell Publishing Ltd. All rights reserved
M.S. Elrobh et al.
66
(a)
TB31
–174.5
+2.5
TB33
–136.5
TB334
–156.5
TB334I-1
In the new constructs described here, efficient repression of the melR promoter by MelR requires interaction
between MelR bound immediately adjacent to the transcript start and upstream-bound MelR, and this can be
subverted by the insertion of a supplementary DNA site
for MelR (Fig. 3). Hence, efficient repression results from
two, but not from three, DNA sites for MelR. Our
experiments underline the diversity of protein–DNA
architectures that can be responsible for transcription
repression.
Acknowledgements
–176.5
TB334I-2
This work was supported by the UK BBSRC with a project grant to S.J.W.B. and a summer studentship to D.D.
(b)
Promoter Fragment
% MelR-dependent Repression
TB31
–MalI
33 (1.1)
+MalI
32 (2.4)
TB33
93 (0.7)
94 (0.7)
TB334
94 (0.4)
93 (0.8)
TB334I-1
92* (0.7)
84* (0.6)
TB334I-2
83* (1.4)
64* (1.9)
Fig. 4. Effects of MalI binding on MelR-dependent repression of the
melR promoter. (a) Schematic diagram of EcoRI-HindIII melR promoter
fragments derived from TB33 with inserted DNA sites for MalI.
Conventions for the different symbols are as in Fig. 1, with open
hexagons representing 16 base pair DNA sites for MalI. (b) The
numbers in the Table denote the% repression of melR promoter
activity in each fragment by MelR, measured as in the legend to
Fig. 1. Measurements were taken in cells that carried either pACYC
malI, encoding malI (+MalI) or empty vector, pACYC-DHN ( MalI).
Each listed result is given as the mean of five independent
determinations, with the standard deviation in brackets. Asterisks
denote data pairs with P-values <0.05 according to the ANOVA
calculator.
at the upstream and downstream DNA targets interact
and result in loop formation, as for AraC. However, there
appears to be more flexibility in how the two DNA sites
for MelR can be juxtaposed, compared to AraC. Hence,
AraC-dependent repression is disrupted by +5 base pair
insertions (Lee & Schleif, 1989), whilst MelR-dependent
repression is not (Fig. 2). The simplest explanation for
this would be that the linker joining the N- and C-terminal domains is more flexible in MelR than in AraC. This
flexibility is underscored by the experiment in Fig. 4
where MalI binding failed to completely disrupt repression. This experiment also argues that the mechanism of
MelR-dependent repression with TB33 is different to the
mechanism operating at the more complex wild type
melibiose operon regulatory region in TB22 (Fig. 1),
where repression depends on the formation of a nucleoprotein complex.
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Published by Blackwell Publishing Ltd. All rights reserved
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1. Complete base sequence of each of the EcoRIHindIII fragments carrying the melR promoter.
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Published by Blackwell Publishing Ltd. All rights reserved