1
Biochem. Soc. Symp. 73, 1–10
(Printed in Great Britain)
 2006 The Biochemical Society
Investigations of the modular
structure of bacterial promoters
Nora S. Miroslavova and Stephen J.W. Busby1
School of Biosciences, The University of Birmingham, Birmingham B15 2TT, U.K.
Abstract
Bacterial RNA polymerase holoenzyme carries different determinants that
contact different promoter DNA sequence elements. These contacts are essential
for the recognition of promoters prior to transcript initiation. Here, we have
investigated how active promoters can be built from different combinations
of elements. Our results show that the contribution of different contacts to
promoter activity is critically dependent on the overall promoter context, and
that certain combinations of contacts can hinder transcription initiation.
Overview
All bacterial genes are transcribed by a multisubunit RNA polymerase that
contains two large subunits, β and β′, and two copies of the smaller α subunit.
Biochemical, genetic and structural studies have shown that the β and β′ subunits
form a cleft that contains the enzyme’s active site for RNA polymerization, and
that the N‑terminal domains of the two α subunits are responsible for holding
the assembly together [1]. Although the RNA polymerase α2ββ′ assembly
(known as the core enzyme) is competent to make RNA, it is unable to initiate
transcription. In order to recognize promoters and to begin transcription,
core RNA polymerase must acquire a σ subunit (resulting in the formation
of RNA polymerase holoenzyme) [2]. All bacteria contain σ subunits, which
function by binding to the surface of RNA polymerase core enzyme, thus
providing determinants for the recognition of specific promoter elements.
Following promoter recognition, the bound σ subunit choreographs a series of
isomerizations that lead to promoter DNA unwinding and entry of the DNA
template into the enzyme active site, where transcript formation is initiated.
Most σ subunits contain four domains that are connected by flexible linkers [3].
Domains 2, 3 and 4 carry determinants for recognition of different promoter
To whom correspondence should be addressed (email [email protected]).
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N.S. Miroslavova and S.J.W. Busby
elements, namely the −10, extended −10 and −35 elements respectively (Figure
1A). Structural studies with Thermus aquaticus RNA polymerase holoenzyme
in complexes with DNA substrates [4,5] have resulted in a clear view of how the
interactions between σ and different promoter elements are organized during
promoter recognition (Figure 1B).
Most bacteria contain a few thousand genes that are expressed from 1000–
2000 promoters. The best‑studied example is E. coli K‑12, which appears typical
of many bacteria, in that the expression of its genes is very tightly regulated,
with changes controlled by alterations in the environment. In most conditions,
a single E. coli cell contains 1000–2000 molecules of RNA polymerase, and
thus effective regulation requires controlled distribution of the polymerase
between the promoters in its genome [6]. This distribution is mainly a result
of the different base sequence determinants found at the promoters, although
an important role is also played by supercoiling and the proteins that interact
with the bacterial chromosome to fold and organize it (these are known as
nucleoid‑associated proteins). Changes in the distribution of RNA polymerase
between promoters are controlled by the production of different σ factors,
and by transcription factors that bind at or near specific promoters and either
activate or repress those promoters in response to specific metabolic signals [7].
Although most studies of the regulation of transcription initiation in E. coli have
focused on promoter sequence determinants, σ factors and transcription factors,
it is now clear that other factors, such as small ligands, small effector proteins
that contact RNA polymerase without contacting promoter DNA, and changes
in the bacterial folded chromosome structure, also play a key role [8,9].
Elements of promoter recognition
In this article, we focus on the primary determinants of promoter activity
in E. coli, which are the initial contacts between RNA polymerase and different
promoter elements. Our study is concerned with RNA polymerase containing
the major σ70 subunit, though the take‑home messages apply to most σ factors.
It has long been known [2,10] that promoter −10 elements are recognized by a
helix in Region 2.4 of σ70 Domain 2, and that a helix–turn–helix in Domain 4 is
responsible for recognition of promoter −35 elements (Figure 1). The consensus
−10 and −35 elements are established as the hexamer sequences TATAAT and
TTGACA respectively. More recent work has shown that a helix in Domain 3 of
σ70 contacts the extended −10 element (consensus TRTGN), located immediately
upstream of the −10 element [3,11]. Finally, the RNA polymerase α subunits play
a key role [12,13]. Each α subunit carries an independently folded C‑terminal
domain (residues 250–329), joined to the α subunit N‑terminal domain by a
flexible linker. The two C‑terminal domains of the two RNA polymerase α
subunits can bind to a 20 bp DNA element, known as the UP element, located at
promoters immediately upstream of the −35 element. The UP element consists
of two half‑elements, each recognized by one α subunit C‑terminal domain
(Figure 1A). A consensus UP element has been determined, and it has been
shown that, at certain promoters, the interactions of RNA polymerase with UP
© 2006 The Biochemical Society
Modular structure of bacterial promoters
Figure 1 RNA polymerase–promoter interactions. (A) The diagram
illustrates the subunit structure of RNA polymerase holoenzyme, showing
the disposition of the different subunits as the enzyme makes contact with
a bacterial promoter. Each α subunit consists of an N‑terminal domain
(αNTD) and a C‑terminal domain (αCTD), joined by a linker. The σ subunit is
divided into four segments to illustrate the four domains. The locations of the
promoter UP, −35, extended −10 and −10 elements are indicated, together
with consensus sequences. The diagram illustrates the different interactions
between RNA polymerase and promoter elements. The small 91‑amino‑acid
ω subunit acts as a chaperone for the β′ subunit [24]. (B) Model of RNA
polymerase holoenzyme docking to promoter DNA, based on structural
studies with Thermus aquaticus RNA polymerase from Seth Darst and
co‑workers [4,5]. [Reproduced with permission from Murakami, K.S., Masuda,
S., Campbell, E.A., Muzzin, O. and Darst, S.A. (2002) Science 296, 1285–1290.
Copyright 2002 AAAS.] The locations of domains 2, 3 and 4 of the σ subunit
(coloured orange) and their interactions with the −10, extended −10 and −35
elements are shown. Note that αCTD and σ domain 1 are not seen in the T.
aquaticus RNA polymerase structure.
© 2006 The Biochemical Society
N.S. Miroslavova and S.J.W. Busby
Figure 2 Building a promoter from different elements. (A) The
complete base sequence of the starting 144 bp NM513 EcoRI–HindIII fragment,
which carries a consensus −10 element (bold type) but non‑consensus
extended −10, −35 and UP elements. The locations of the different elements
are indicated by horizontal lines. The transcription start site, +1, is in bold
and underlined. (B) Partial base sequences of the starting NM513 promoter
and three derivatives, carrying different promoter elements, are shown from
positions −55 to +1. NM535 carries consensus −35 and −10 elements, shown
in bold type. NM511 carries consensus extended −10 and −10 elements (bold).
NM501 carries a consensus −10 element and a DNA site for CRP (bold). All
the promoters carry identical sequences downstream of the transcription
start site. Each promoter was fused to the lacZ gene (in the expression
vector pRW50 [18]), and its ability to initiate transcription was deduced from
measurements of β‑galactosidase activities in the ∆lac E. coli strain MC4100
growing exponentially. Cells were grown at 37°C in Luria–Bertani medium,
supplemented with tetracycline (35 µg/ml). Activities are expressed as nmol of
o‑nitrophenyl β‑d‑galactoside hydrolysed⋅min−1⋅mg dry cell mass−1 (Miller units)
and are listed in the final column. (C) Partial base sequence from positions
−55 to +1 of the NM500 promoter, which carries a DNA site for CRP, and
consensus extended −10 and −10 elements (shown in bold). Expression from
this promoter was deduced from the β‑galactosidase activity measurement
shown in the final column.
© 2006 The Biochemical Society
Modular structure of bacterial promoters
Figure 3 Reduction of promoter activity due to too many consensus
promoter elements. (A) Partial base sequences from positions −50 to
+1 of four promoters containing a consensus −10 element, with or without
a consensus −35 hexamer, and with or without a consensus extended −10
element. Consensus sequence elements are indicated by bold type. Each
promoter was fused to the lacZ gene, and its ability to initiate transcription was
deduced from measurements of β‑galactosidase activities in a ∆crp derivative of
E. coli strain MC4100, growing exponentially in Luria–Bertani medium. Activities
are expressed as nmol of o‑nitrophenyl β‑d‑galactoside hydrolysed⋅min−1⋅mg
dry cell mass −1 (Miller units) and are listed in the final column. (B) Derivatives
of the four promoters in (A) in which the consensus −10 hexamer, TATAAT,
was replaced with TATGGT. Activities were assayed exactly as in (A).
element sequences can improve promoter activity by a factor of 10‑fold or much
more [14,15].
Our strategy was to investigate how different combinations of promoter
elements could make functional promoters. The starting point was the EcoRI–
HindIII fragment illustrated in Figure 2(A) that carries the NM513 promoter.
This fragment was derived from the E. coli galactose operon promoter region
[16] which had been engineered to carry a consensus −10 hexamer element,
TATAAT, but no consensus extended −10 or −35 element and no UP element,
so that it possessed little or no promoter activity. Our aim was to introduce
© 2006 The Biochemical Society
N.S. Miroslavova and S.J.W. Busby
different combinations of promoter elements and to measure the resulting
promoter activities. Figure 2(B) illustrates the NM535 promoter, with an added
consensus −35 element, and the NM511 promoter, with a consensus extended
−10 element. We also constructed the NM501 promoter, which carries a DNA
site for the transcription activator CRP (cAMP receptor protein). The reason
for this was to show that promoter activity could be created by the intervention
of trans‑acting factors as well as by the addition of cis‑acting promoter elements.
CRP is a well understood bacterial transcription activator [17], and thus we
placed a DNA site for CRP at a location where bound CRP would be competent
to activate transcription.
To measure the activity of the new promoter constructions, the different
EcoRI–HindIII fragments were transferred to plasmid pRW50, a promoter‑probe
vector that places the lac genes under the control of cloned promoters [18].
The resulting pRW50 derivatives were then transformed into the ∆lac E. coli
K‑12 strain MC4100, and β‑galactosidase activities were measured as an index
of the activity of the cloned promoter (as described in [18]). Data in Figure
2(B) show that, as expected, the starting NM513 promoter had very low activity
(expression was close to background levels), whereas the presence of a consensus
−35 element (in NM535), an extended −10 element (in NM511) or bound CRP
(in NM501) created promoter activity.
Promoter activity: more can mean less
Figure 2(C) illustrates the NM500 derivative of the NM513 promoter, which
carries both an extended −10 element and a DNA site for CRP. The activity of the
NM500 promoter is greater than the individual activities of the NM511 and NM501
promoters, showing that an extended −10 element can work together with CRP.
Figure 3(A) illustrates an experiment to investigate whether, in the context of
a promoter with a consensus −10 element, an extended −10 element can function
positively with a consensus −35 element. The data show that the addition of an
extended −10 element to a −35 element in the NM530 promoter caused a decrease
in promoter activity. Thus we conclude that too many strong contacts between a
promoter and RNA polymerase may hinder rather than help a promoter, presumably
because the bound RNA polymerase becomes anchored at the promoter and is less
able to escape. Support for this point of view comes from the experiment illustrated
in Figure 3(B), in which the −10 element was altered from the consensus, TATAAT,
to TATGGT. Thus the activity of the NM130 promoter carrying both a consensus
−35 element and an extended −10 element was higher than the activities of the
NM131 or NM100 promoters that carry either one element or the other.
UP element contributions to promoter activity are
contingent on the −35 element
In the promoters illustrated in Figure 2, the DNA sequence just upstream
of the −35 region was chosen so as not to resemble any known UP element.
© 2006 The Biochemical Society
Modular structure of bacterial promoters
Indeed, in the starting NM513 promoter, we used a DNA sequence that had
been characterized previously as an ‘anti‑UP’ sequence, i.e. the sequence least
likely to enhance promoter activity due to binding of the C‑terminal domain
of the RNA polymerase α subunit [15]. In order to measure the effects of
introducing a UP element, a 20 bp sequence, selected previously for its optimal
UP element function [15], was cloned upstream of the −35 region at the NM511
or NM535 promoters (Figure 4). This yielded the NM510 promoter (carrying
a UP element, and consensus −10 and extended −10 elements) and the NM534
promoter (carrying a UP element, and consensus −10 and −35 elements). To
measure the effects of these UP elements, DNA fragments carrying the NM510
and NM534 promoters were cloned into the pRW50 lac expression vector as
before. The data in Figure 4 show that the UP elements had small effects on
promoter activity at these promoters.
We reasoned that the effects of the UP elements at the NM510 and NM534
promoters might be increased if the other promoter elements were weakened.
Thus we made a series of derivatives of NM534 in which the consensus −35
element was systematically mutated. These changes are illustrated in Figure 5;
positions 2–5 of the consensus −35 hexamer, TTGACA, were changed to each
of the three alternative bases (note that position 1 was not altered, as we wished
to retain the BglII restriction site immediately upstream of the −35 element).
The same set of 15 mutations was also created in the context of the NM535
promoter, which lacked a UP element. Figure 5 shows a comparison of the
effects of each base change on promoter activity in either NM534 or NM535.
Two clear results emerged. First, at the NM535 promoter without a UP element,
the effects of the different −35 hexamer changes were in accordance with those
reported previously in similar studies with the E. coli lacUV5 promoter [19] and
Figure 4 Effects of a UP element at different promoters. Partial
base sequences of promoters containing or lacking UP element sequences in
combination with either consensus extended −10 and −10 elements (NM511
and NM510) or consensus −35 and −10 elements (NM535 and NM534) are
shown. Consensus sequence elements are indicated by bold type. All of the
promoters carry identical sequences downstream of the transcription start site.
Each promoter was fused to the lacZ gene, and its ability to initiate transcription
was deduced from measurements of β‑galactosidase activities in the ∆lac E. coli
strain MC4100 growing exponentially in Luria–Bertani medium. Activities are
expressed as nmol of o‑nitrophenyl β‑d‑galactoside hydrolysed⋅min−1⋅mg dry
cell mass−1 (Miller units), and are listed in the final column.
© 2006 The Biochemical Society
N.S. Miroslavova and S.J.W. Busby
Figure 5 Base changes in the promoter −35 element affect UP
element function. The bar chart shows measured b‑galactosidase activities
during exponential growth of E. coli MC4100 ∆lac cells containing pRW50
plasmid carrying lac fusions to the NM535 promoter and its derivatives (no
UP element; black bars) or to the NM534 promoter and its derivatives (with
a UP element; grey bars). The derivatives of both promoters carry single base
changes at positions 2–6 of the consensus −35 element, TTGACA, as indicated
under the bar chart; the location of each substitution is underlined. Assays
were performed as in Figure 2. Activities are expressed as a percentage of the
activity with the starting NM535 promoter (100%; equivalent to 625 Miller
units). Values are means+S.E.M. for three independent experiments.
the Salmonella P22 ant promoter [20]. Thus different changes reduce promoter
activities by different amounts. Secondly, the presence of the UP element
resulted in larger increases in promoter activity at the promoters where the −35
elements caused the biggest decreases. Hence it is clear that the UP element can
compensate for defects in the −35 element.
To explore further the idea that the effects of the UP element are greater
at weaker promoters, mutations that altered the consensus −10 element from
TATAAT to CATAAT or TGTAAT were made in the NM534 and NM535
promoters carrying a consensus −35 element, TTGACA, and derivatives in
which the −35 element was changed to either TCGACA or TTGGCA. The
results illustrated in Figure 6 show that UP‑element‑dependent stimulation of
promoter activity is greater at promoters that have a defective −35 element than
at promoters with a defective −10 element.
Conclusions
The recognition of promoters by the multisubunit bacterial RNA polymerase
involves four different protein determinants that recognize four different promoter
elements. Our results show that an efficient promoter can be built by different
© 2006 The Biochemical Society
Modular structure of bacterial promoters
Figure 6 Effects of −10 and −35 element sequences on UP element
function. The bar chart shows measured β‑galactosidase activities during
exponential growth of E. coli MC4100 ∆lac cells containing pRW50 plasmid
carrying lac fusions to the NM535 promoter and its derivatives (no UP element;
black bars) or to the NM534 promoter and its derivatives (with a UP element;
grey bars). The derivatives of both promoters carry combinations of the −35
hexamer elements TTGACA, TCGACA and TTGGCA and the −10 hexamer
elements TATAAT, CATAAT and TGTAAT, as indicated. The location of each
substitution is underlined. Assays were performed as described in Figure 2.
Activities are expressed as a percentage of the activity with the starting NM535
promoter (100%; equivalent to 6251 Miller units). Values are means+S.E.M. for
three independent experiments.
combinations of these contacts. It is also clear that promoter activity reaches
a ceiling if too many contacts are created (see also [21]). Presumably there is a
trade‑off between contacts being sufficiently strong for promoter binding, but
not too strong so that promoter escape is hindered. Our results suggest that, to
some extent, the −35 element and the UP element are redundant. Presumably this is
because they are both involved in the very initial contacts between a target promoter
and RNA polymerase [22]. Hence there is greater synergy between the UP element
and the promoter −10 element. Interestingly, all naturally occurring promoters use
sub‑optimal combinations of the different contacts [23]. There appear to be two
reasons for this. First, because expression levels of genes need to be controlled, the
‘tuning’ of a promoter’s activity is more important than the optimization of its
activity. Secondly, sub‑optimally active promoters can, if needed, be activated by
transcription factors. Hence, in E. coli (and probably also in other organisms), the
government of the cell favours modest promoters and rejects greedy ones!
We thank the Wellcome Trust for funding this work with a project grant, and the
Darwin Trust of Edinburgh for supporting N.M.’s Ph.D. programme. Automated
DNA sequencing was performed by the University of Birmingham Functional
Genomics Laboratory, supported by grant 6/JIF13209 from the U.K. Joint
Infrastructure Fund.
© 2006 The Biochemical Society
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N.S. Miroslavova and S.J.W. Busby
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© 2006 The Biochemical Society