Mechanism of promoter repression by Lac repressor–DNA loops
ABSTRACT The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repres-sor concentration come from free repressor and re-pressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.
- SourceAvailable from: Peter Lindblad[Show abstract] [Hide abstract]
ABSTRACT: Cyanobacteria are solar-powered prokaryotes useful for sustainable production of valuable molecules, but orthogonal and regulated promoters are lacking. The Lac repressor (LacI) from Escherichia coli is a well-studied transcription factor that is orthogonal to cyanobacteria and represses transcription by binding a primary lac operator (lacO), blocking RNA-polymerase. Repression can be enhanced through DNA-looping, when a LacI-tetramer binds two spatially separated lacO and loops the DNA. Ptrc is a commonly used LacI-repressed promoter that is inefficiently repressed in the cyanobacterium Synechocystis PCC 6803. Ptrc2O, a version of Ptrc with two lacO, is more efficiently repressed, indicating DNA-looping. To investigate the inefficient repression of Ptrc and cyanobacterial DNA-looping, we designed a Ptrc-derived promoter library consisting of single lacO promoters, including a version of Ptrc with a stronger lacO (Ptrc1O-proximal), and dual lacO promoters with varying inter-lacO distances (the Ptrc2O-library). We first characterized artificial constitutive promoters and used one for engineering a LacI-expressing strain of Synechocystis. Using this strain, we observed that Ptrc1O-proximal is similar to Ptrc in being inefficiently repressed. Further, the Ptrc2O-library displays a periodic repression pattern that remains for both non- and induced conditions and decreases with longer inter-lacO distances, in both E. coli and Synechocystis. Repression of Ptrc2O-library promoters with operators out of phase is less efficient in Synechocystis than in E. coli, whereas repression of promoters with lacO in phase is efficient even under induced conditions in Synechocystis. Two well-repressed Ptrc2O promoters were highly active when tested in absence of LacI in Synechocystis. The artificial constitutive promoters herein characterized can be utilized for expression in cyanobacteria, as demonstrated for LacI. The inefficient repression of Ptrc and Ptrc1O-proximal in Synechocystis, as compared to E. coli, may be due to insufficient LacI expression, or differences in RNAP subunits. DNA-looping works as a transcriptional regulation mechanism similarly as in E. coli. DNA-looping contributes strongly to Ptrc2O-library repression in Synechocystis, even though they contain the weakly-repressed primary lacO of Ptrc1O-proximal and relatively low levels of LacI/cell. Hence, Synechocystis RNAP may be more sensitive to DNA-looping than E. coli RNAP, and/or the chromatin torsion resistance could be lower. Two strong and highly repressed Ptrc2O promoters could be used without induction, or together with an unstable LacI.Journal of Biological Engineering 01/2014; 8(1):4.
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ABSTRACT: Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor.PLoS ONE 01/2014; 9(5):e92475. · 3.53 Impact Factor
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ABSTRACT: The Escherichia coli lactose operon provides a paradigm for understanding gene control by DNA looping where the lac repressor (LacI) protein competes with RNA polymerase for DNA binding. Not all promoter loops involve direct competition between repressor and RNA polymerase. This raises the possibility that positioning a promoter within a tightly constrained DNA loop is repressive per se, an idea that has previously only been considered in vitro. Here, we engineer living E. coli bacteria to measure repression due to promoter positioning within such a tightly constrained DNA loop in the absence of protein-protein binding competition. We show that promoters held within such DNA loops are repressed ∼100-fold, with up to an additional ∼10-fold repression (∼1000-fold total) dependent on topological positioning of the promoter on the inner or outer face of the DNA loop. Chromatin immunoprecipitation data suggest that repression involves inhibition of both RNA polymerase initiation and elongation. These in vivo results show that gene repression can result from tightly looping promoter DNA even in the absence of direct competition between repressor and RNA polymerase binding.Nucleic Acids Research 03/2014; · 8.81 Impact Factor
Mechanism of promoter repression by Lac
Nicole A. Becker1, Justin P. Peters1, Troy A. Lionberger2and L. James Maher III1,*
1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street
Southwest, Rochester, MN 55905, USA and2Howard Hughes Medical Institute and Jason L. Choy Laboratory
of Single-Molecule Biophysics, Department of Physics, University of California, Berkeley, CA 94720, USA
Received June 29, 2012; Revised October 1, 2012; Accepted October 2, 2012
The Escherichia coli lactose (lac) operon encodes
the first genetic switch to be discovered, and lac
remains a paradigm for studying negative and
control is believed to involve competition of RNA
polymerase and Lac repressor for overlapping
binding sites. Contributions to the local Lac repres-
sor concentration come from free repressor and re-
pressor delivered to the operator from remote
auxiliary operators by DNA looping. Long-standing
questions persist concerning the actual role of DNA
looping in the mechanism of promoter repression.
Here, we use experiments in living bacteria to
resolve four of these questions. We show that the
distance dependence of repression enhancement is
comparable for upstream and downstream auxiliary
operators, confirming the hypothesis that repressor
concentration increase is the principal mechanism
of repression loops. We find that as few as four
turns of DNA can be constrained in a stable loop
by Lac repressor. We show that RNA polymerase
is not trapped at repressed promoters. Finally, we
show that constraining a promoter in a tight DNA
promoter and operator do not overlap.
It is difficult to overstate the historical significance of the
Escherichia coli lactose (lac) operon as a paradigm for
negative and positive control of gene expression (1). This
short segment of the bacterial chromosome encodes a
genetic switch that senses and responds to glucose and
lactose concentrations to produce lactose digestion enzymes
only when glucose is absent and lactose is present (2).
Central to this function is the homotetrameric Lac repressor
protein that binds DNA operator sequences in the lac
operon (3). Lac repressor binding is weakened in the
presence of allolactose or its analog, isopropyl b-D-1-
thiogalactopyranoside (IPTG), relieving repression. In the
absence of glucose, RNA polymerase binds cooperatively
control). In simplest terms, the mechanism of negative
control involves Lac repressor binding to occlude access
of RNA polymerase holoenzyme to the lac promoter (4).
Of particular significance to the present work is the
fascinating observation that two remote auxiliary oper-
ators (Oaux) exist in the lac operon (5). It has been
proposed and demonstrated (6–13) that bidentate repres-
sor tetramers bound at auxiliary operators increase the
operator (O) through DNA looping (Figure 1). This
concept has been exploited as an approach to probe the
physical properties of bacterial DNA in vitro (14) and
within the bacterial nucleoid in vivo (12,15–17).
The natural auxiliary operators of the lac operon have
different affinities and occur such that one is just upstream
and one is far downstream of the regulatory operator im-
mediately proximal to the promoter. We have designed
experiments in living bacteria to clarify the role of DNA
looping in repression. We first tested the hypothesis that
increasing the local concentration of repressor at the
proximal operator is necessary and sufficient to explain
promoter repression (13). This hypothesis implies that
identical auxiliary operators positioned equivalent dis-
tances either upstream or downstream of the regulatory
operator should provide comparable enhancement of
repression by equally increasing the effective local repres-
sor concentration at the regulatory operator. Artificial lac
operators have been shown to enhance repression when
upstream or downstream of the promoter (18), but the
equivalence of upstream and downstream auxiliary
operators has not been previously tested in systematic ex-
periments. Second, we determined the smallest possible
DNA loop involving Lac repressor in living bacteria.
Third, we tested the early proposal that favorable
repressor-polymerase contacts trap RNA polymerase at
repressed promoters (19,20). Fourth, we tested in vivo a
recent hypothesis that promoter DNA bending strain is
intrinsically repressive (21).
*To whom correspondence should be addressed. Tel: +1 507 284 9041/98; Fax: +1 507 284 2053; Email: firstname.lastname@example.org
Nucleic Acids Research, 2013, Vol. 41, No. 1 Published online 9 November 2012
? The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which
permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
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MATERIALS AND METHODS
FW102 (the kind gift from F. Whipple) is a streptomycin
resistant derivative of CSH142 [araD(gpt-lac)5] and is
designated as wild-type in this study (22). Gene deletions
and the presence of looping assay episomes were con-
firmed by diagnostic polymerase chain reaction (PCR)
amplification after conjugation and selection (23).
DNA looping constructs were based on plasmid pJ992,
created by modifications of pFW11-null (22) as previously
described (23). The relationship between lac operator
sequence and repression in the absence of DNA looping
were measured in preliminary experiments (Supplemen-
tary Figure S1). Sequences of new experimental and
control promoters are shown in Supplementary Figure
S2 with descriptions in Supplementary Table S1. The O2
operator normally present within the lacZ coding region
was destroyed by site-directed mutagenesis (15). The
experimental strong UV5 promoter does not contain a
catabolite activator protein binding site. lacZ looping con-
structs were placed on the single copy F128 episome by
homologous recombination between the constructed
plasmids and bacterial episome followed by mating.
F128 carries the lacI gene producing wild-type levels of
repressor. Bacterial conjugation and selections were as
previously described (23).
In vivo DNA looping assay and data fitting
Analysis of lac reporter gene expression was performed as
described (23). Raw b-galactosidase reporter activity (E) is
presented in Miller units. Normalized E0values are then
obtained by dividing E values by E obtained for a test
construct where specific looping is not possible because
only a single proximal O2 operator is present in the
absence of an auxiliary operator. The repression ratio
(‘RR’) is given by Einduced/Erepressed, where induction is
obtained by addition of 2mM IPTG. Best fits to the
thermodynamic model of lac gene regulation produced
estimates of seven parameters (Table 1) as described
(23). The LacI Y282D mutation was created using
QuikChange Lightning Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA, USA). Upper and lower
mutagenic primers were LJM-4110 50-GACGATAC2
TCA3CAG and LJM-4111 50-CTGT3GATG2TG2T2A2
with the mutation underlined. LacI Y282D mutant
looping constructs were placed on the single-copy F128
episome by homologous recombination. After mating
and selection, correct strain recombinants were confirmed
by PCR amplification to detect the inactivated internal
lacZ O2sequence with PCR primers LJM-1930 (50-CGT
CGT4ACA2CGTCG) and LJM-1931 (50-CAT2GA3GT2
A2TGA2TAGCAC). The LacI Y282D mutation was
confirmed by PCR amplification followed by PCR
product sequencing using primers LJM-4112 (50-A2G2C
GACTG2AGTGC2ATG) and LJM-4113 (50-GA3C2TGT
CGTGC2AGCTG). All data are included in Sup-
plementary Table S2. For studies of T7 RNA polymerase
promoters, promoter–reporter constructs were created
based on plasmid pJ992, with modifications illustrated in
Supplementary Figure S4, and were placed on the single-
copy F128 episome. Cells were transformed with a
plasmid expressing an arabinose inducible T7 RNA poly-
merase gene (a kind gift from Troy A. Lionberger) before
lac reporter gene analysis. To determine b-galactosidase
activity, bacterial cultures were grown in MOPS minimal
buffered media (Teknova) supplemented with 0.8% of
10mM of NaHCO3, 0.2% of casamino acids and
12.5mg/ml of thiamine. Cultures were grown either in
the presence or absence of 2mM IPTG and/or 0.02% ara-
described (23). T7 RNA polymerase promoter–reporter
constructs are illustrated schematically in Supplementary
Figure S4, with data shown in Supplementary Table S3.
Escherichia coli cultures were grown to log phase in 50 ml
cultures of LB medium at 37?C in the presence or absence
of 2mM IPTG. Cross-linking of DNA and protein
complexes was accomplished with the addition of 37%
formaldehyde (Sigma) to make a final concentration of
1% in the presence of 10mM sodium phosphate (pH
7.6). Cultures were maintained at room temperature
with constant gentle swirling for 20min. Reactions were
quenched with cold 2M glycine (200mM of final concen-
tration). Cells were harvested by centrifugation, washed
three times with 4 ml of cold phosphate buffered saline
and were resuspended in 1 ml of IP buffer [100mM of
Tris–HCl pH 8.0, 300mM of NaCl, 2% of Triton
X-100, 1mM of PMSF and an additional protease inhibi-
tor mix (Roche)]. Cells were lysed, and cellular DNA was
sheared by sonication and was further analysed as
described in the Supplementary Materials.
RESULTS AND DISCUSSION
The experimental design for this work is illustrated in
Figure 2. We have previously studied a series of artificial
lac UV5 promoters, where repression by a weak proximal
operator (O2) is dramatically enhanced in a distance-
dependent manner by a strong auxiliary operator (Osym)
Figure 1. Hypothetical contributions to local bidentate repressor con-
centration at a bacterial operator (O) include contributions because of
free repressor and contributions because of DNA-bound repressor at
auxiliary operators (Oaux) through DNA looping.
Nucleic AcidsResearch, 2013, Vol.41,No. 1 157
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(15–17,24,25). Repression loops formed by Lac repressor
trap the promoter within a tightly bent segment, where O2
occupancy by repressor is assumed to occlude polymerase
binding (dashed region in Figure 2B). We now create a
series of promoter constructs illustrated in Figure 2C (see
Supplementary Figure S2). In these constructs, the same
promoter and proximal O2 elements are supplemented
with the auxiliary Osymoperator positioned ‘downstream’
such that looping again enhances repressor occupancy at
O2but does so without constraining the promoter within
the loop (Figure 2D). Effects of Osymoperators studied in
the absence of DNA looping are shown in Supplementary
Figure S3. The hypothesis to be tested is that promoter
repression is equivalent for DNA loops of comparable
size, regardless of whether the regulated promoter lies
within the loop (Figure 2B) or adjacent to the loop
(Figure 2D); ‘it is only the DNA loop-dependent
increase in effective Lac repressor concentration at O2
that determines repression’. The particular alternative
hypothesis of interest is that promoters entrapped within
strained DNA loops (Figure 2B) are repressed more
completely than when the promoter is not strained
upstream ofthepromoter (Figure2A)
Upstream and downstream loops in control studies
Experiments were undertaken byplacing aseries ofspacing
constructs (Supplementary Figure S2 and Table S2) in a
single copy on the F0episome of E. coli and measuring
reporter gene (lacZ) expression in the absence or presence
of IPTG inducer. Data were collected as a function of
operator spacing and were fit to a thermodynamic model
to estimate parameters describing the physical properties
of the intervening DNA (12,15,23). We began with a set
of control experiments to assess the effect of DNA loops
formed upstream (Figure 3A and B) or downstream
(Figure 3C and D) of the proximal O2operator. The data
in Figure 3 show how the promoter RR (induced reporter
expression divided by repressed reporter expression)
depends on the number and location of operators. Given
that the apparent DNA helical repeat for the supercoiled
lac promoter region in vivo is between 10.8 and 11bp/turn
(25), for an upstream Oaux, with O2and Osymoperators
separated by 65bp (center-to-center), the operators are
on the same DNA face, yielding strong control and an
RR value of 99 (Figure 3A). Repression by occupancy of
Table 1. Parameters (95% confidence interval) fit to a thermodynamic model of Lac repressor looping
Parameters not well determined by fitting are indicated in italics.
aFits based on data reported by Becker et al. (2005).
bFits based on current data set after eliminating data for spacings <41.5bp.
cFits based on downstream data set with mutant LacI after eliminating data for spacings <41.5bp.
Figure 2. Promoter–reporter constructs to compare repression by
upstream (A and B) versus downstream (C and D) DNA loops in the
presence (A and C) or absence (B and D) of IPTG inducer. The dashed
region indicates the RNA polymerase footprint, emphasizing that re-
pressor binding at O2occludes promoter access.
158Nucleic Acids Research, 2013,Vol. 41,No. 1
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the weak O2operator without DNA looping drops by
>30-fold, and no repression is detectable when O2is dis-
rupted (Figure 3A). Results are similar, but the strength of
repression is reduced to ?4-fold, for operators spaced by
73bp (Figure 3B). Energetically unfavourable DNA
twisting is required for closure of the repression loop in
this case. We then tested the construct shown in Figure
3C, where the relationship of the lac UV5 promoter and
proximal O2operator is unchanged, but the auxiliary Osym
operator is placed downstream. For an operator spacing
(67bp centre-to-centre) that places repressors on the same
DNA face, repression is again seen to be strong (RR value
68) and dependent on the presence of both operators
(Figure 3C). Interestingly, in the absence of DNA
looping, the presence of Osym ?50–90bp downstream
from the promoter yields RR values of 4–9 (Figure 3C
and D). These results suggest that a tightly bound Lac
repressor interferes weakly, but detectably, with RNA
polymerase elongation (18,26). Loop-dependent repres-
sion remains detectable, but weaker, for an unfavorable
Importantly, loops involving an O2operator positioned
20 or 40bp downstream from the promoter were no more
inhibitory than isolated repressor–operator complexes at
these locations (Figure 3D). This result demonstrates that
enhanced repression in experiments where Osymis down-
stream of the promoter depends on O2adjacent to the
promoter. This result also suggests that like upstream
(50bp, Figure 3D).
represses through inhibition of transcription initiation.
This demonstrated similarity in mechanism allows direct
comparison of quantitative data from upstream and down-
Length-dependent upstream and downstream lac loop
We have previously studied apparent DNA physical
properties in living bacteria by monitoring promoter repres-
sion as a function of operator spacing for upstream loops
(Figure 2A and B). We reported an oscillating pattern of
repression as a function of operator spacing (Figure 4A and
B) consistent with unexpectedly low DNA bending resist-
ance, but strong DNA twist resistance, residual looping
even in the presence of IPTG and a deduced DNA helical
repeat parameter that differed between repressing and
inducing conditions (15–17,24,25). New constructs with
downstream auxiliary operators were studied, and the
data are shown in Figure 4C and D with fitting to a
thermodynamic model of DNA looping (Table 1).
Figure 4C shows reporter activity normalized to a construct
with only a proximal operator (E0) as a function of operator
spacing (center-to-center). In Figure 4C, data for both re-
pressing (filled black circles, solid red fit curve) and
inducing (open black circles, dashed red fit curve) condi-
tions are compared with data for loops involving upstream
auxiliary operators (shaded region). The pattern is similar,
although repressed E0values tend to be lower for constructs
with downstream auxiliary operators. We noted that oscil-
lations in repressed and induced E0values are somewhat
irregular for operator spacings between 40 and 70bp
(Figure 4C), whereas oscillation of their RR (filled black
circles and red line fit in Figure 4D) is regular and similar to
the result for upstream loops (Figure 4D, solid grey curve).
We interpret the irregular oscillation of downstream
E0values (Figure 4C) and regular oscillation of RR
values (Figure 4D) as evidence of mRNA sequence-
dependent gene expression for downstream auxiliary oper-
ators; sequence changes involving downstream auxiliary
leaders that may affect RNA stability and/or translation
rate. Normalization removes such effects in the RR data
(Figure 4D). Also different from results for upstream aux-
iliary operators in the absence of proximal operators
(Figure 4B, filled triangles and dashed curve), constructs
carrying a strong downstream auxiliary Osymoperator in
the absence of a proximal O2operator show oscillating
phase-dependent repression (Figure 4D, filled triangles,
dashed black curve). This loop-independent repression is
higher than for upstream auxiliary operators (Figure 4D,
dashed grey line fit), again suggesting that an occupied Osym
represents a barrier to transcription elongation by E. coli
RNA polymerase. This oscillation could reflect Osym
sequence effects or residual DNA looping anchored by
weak recognition of the disabled proximal O2operator or
some other unknown cryptic operator sequence (10).
To resolve this issue, we reproduced the study of down-
stream Osym effects in an E. coli strain with a totally
disabled Lac repressor [LacI Y282D (27–29)]. The
Figure 3. RR behavior of the indicated promoter–reporter constructs.
UV5 promoter elements (?35, ?10), center-to-center spacing of weak
and strong lac operators (O2and Osym, respectively), Shine-Dalgarno
(sd) and reporter (lacZ) are shown, as well as the length of any insert
between the transcription start site (broken arrow) and proximal O2
operator. Boxed data show RR values for the indicated operator com-
binations, where filled black rectangles indicate intact operators and
open slashed rectangles indicate disrupted operators. RR values in
bold highlight comparisons. (A and B) Upstream loops with operators
in phase (A) or out of phase (B). (C and D) Downstream loops with
operators in phase (C) or out of phase (D).
Nucleic AcidsResearch, 2013, Vol.41,No. 1 159
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results are shown in Figure 4 (panels E and F). The E0
expression data (Figure 4, panel E, compare green to
dashed red) show a comparable oscillation in the
complete absence of Lac repressor relative to the
original (dashed red) induced (+IPTG) E0data. In panel
F of Figure 4, these RRs data (green) do not oscillate,
confirming the absence of active repressor. This result in-
dicates that the residual E0oscillation (green in Figure 4E)
is because of some feature of the Osymoperator sequence,
as it rotates about the helix axis, ‘not’ residual weak
looping because of weak repressor binding in the
presence of IPTG. Given that the effect of the downstream
Osymremains even in the complete absence of repressor,
the origin of the oscillating pattern is mysterious and inter-
esting. Oscillation with the helical repeat of DNA implies
a face-of-the-helix effect. Possible explanations could
include interactions between the promoter and a DNA
structure at the strong inverted repeat at Osym or a
strong hairpin structure in the nascent RNA transcript
or even weak binding by some other protein at Osym.
Thus, there is evidence for an oscillating effect of the pal-
indromic operator sequence, per se, on gene expression.
The data in the complete absence of functional Lac re-
pressor also provide the opportunity to express the RR as
fully induced (no repressor) divided by fully repressed.
These data are included in Figure 4F (magenta) and
show the much larger dynamic control range when
residual repression looping is eliminated, as previously
The effect of isolated auxiliary Osymwas also measured
in the absence of either a proximal operator or residual
repressor (LacI Y282D mutant). This is shown in panels C
and D of Supplementary Figure S3. Supplementary Figure
S3C (light solid grey) shows weak oscillation previously
observed for upstream Osymauxiliary operators, suggest-
ing little position effect of upstream Osym, as reported (15).
In Supplementary Figure S3C, the E0(green) data in the
absence of functional repressor show that isolated Osym
oscillating repression effect that is similar to the wild-type
LacI induced by IPTG, indicating that there is an
oscillating effect of the Osym operator sequence alone,
that is magnified when Lac repressor binds to it. Supple-
mentary Figure S3D (RR) shows that repressor binding to
the isolated downstream Osymoperator amplifies the effect
of the operator (compare bLack dashed trace with green
It is important to note that loop-independent repression
by a transcribed downstream Osymoperator can produce
RR values of 4–9 (Figure 4D, filled triangles), equivalent
to cases where a proximal O2operator and downstream
Osymoperator are out of phase. This repression in the
absence of looping is because of collisions between repres-
Figure 4. Repression data and fits to thermodynamic model for loops with upstream (A and B) versus downstream (C–F) auxiliary Osymoperators.
Reporter activity is shown as E’ (A, C and E) where the shaded envelope in (A) indicates behavior of upstream loop constructs under induced (open
symbols, dashed fit curve) or repressed (filled symbols, solid fit curve) conditions with fits to thermodynamic model. Blue symbols for spacings closer
than the vertical line at 41bp (C–F) indicate constructs that do not show a canonical looping pattern. Panels (B, D and F) show RR data and fits to
the thermodynamic model (see Supplementary Figure S3). Filled triangles and dashed fits indicate RR data for strains containing only an auxiliary
Osymoperator in the absence of a proximal operator. Grey fits in panels (D) and (F) indicate RR behaviour of upstream loops from (B). Green
symbols and fits in panels (E) and (F) show data obtained in the absence of functional Lac repressor (LacI Y282D). Data and fit in magenta (panel
F) show the modified RR for downstream loop constructs where the numerator reflects reporter expression in the absence of functional repressor
(LacI Y282D) and the denominator reflects repressed reporter expression in the presence of wild-type Lac repressor.
160 Nucleic Acids Research, 2013,Vol. 41,No. 1
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polymerase. This means that the 68-fold repression for the
downstream loop observed in Figure 3C is only 7–14-fold
higher than for the isolated Osymoperator (compared with
the 33-fold ratio related to isolated Osymfor the upstream
loop in Figure 3A). However, compared with O2alone,
the upstream RR increases by 33-fold and downstream by
a similar 23-fold.
By fitting to a thermodynamic model of the Lac control
loop (23), these data produce estimates for the seven par-
ameters given in Table 1. Most striking is the difference in
fit values for the apparent loop twist constant, Capp, whose
estimates are >3-fold higher for downstream loops than
for upstream loops. Because a common value for the
DNA twist constant in vitro is ?2.4?10?19erg-cm, this
suggests that it is the twist flexibility of the upstream loop
that is anomalously high, even given the fact that the Lac
repressor protein constitutes part of the loop. Because this
parameter is dominated by the behavior of the UV5
promoter sequence in the upstream loops, it is possible
that this result reflects a relatively high twist flexibility
for the promoter sequence. In this regard, it has been pre-
viously shown by DNaseI accessibility (31) and cyclization
kinetics (32) that TATA-like sequences display anomalous
mechanical anisotropic flexibility that might explain our
observations. Also noteworthy is the 5-fold higher fit
value of K0max (the equilibrium constant for loops
without twisting strain) for downstream loops. It is
unclear why optimal downstream loops are more energet-
ically favorable (as implied by this value). It is again
possible that unique aspects of the Lac UV5 promoter
could be reflected in this parameter.
Smallest detectable Lac repression loop in vivo
The design of Lac loop constructs with an auxiliary Osym
operator downstream of O2(Figure 2C and D) provided
an opportunity to identify the smallest possible Lac re-
pression loop. In previous work with upstream loops, we
minimized the spacing of operators flanking an E. coli
promoter until interference was detected (25). For oper-
ators flanking an E. coli promoter, the smallest centre-to-
centre operator spacing for a repression loop was ?60bp
(25). Examination of the data in Figure 4C and D shows
that DNA looping fails between 40 and 45bp (about four
turns of the double helix, two of which are between the
operators). For operator spacings <40bp (center-to-
center), there is no evidence of loop-dependent promoter
repression (Figure 4C and D, blue symbols). This suggests
that Lac repressor can constrain four turns of DNA into a
stable loop. Interestingly, Hochschild and Ptashne (33)
previously reported that loops driven by protein–protein
interactions between cooperatively bound of phage ? re-
pressor dimers could be detected for center-to-center
spacings of as small as five DNA turns. Thus, the
flexible tetrameric Lac repressor seems to be capable of
stabilizing more highly strained DNA loops than phage ?
repressor. Such tight DNA looping is likely to be
facilitated by the high affinity of Lac repressor, its stable
tetrameric structure and the flexibility of the Lac repressor
tetramer, which has the potential to form an extended
linear structure in bridging operators within a micro-
Fate of RNA polymerase at Lac repression loops in vivo
The data in Figures 3 and 4 and Table 1 support the hy-
pothesis that promoter repression is principally by binding
occlusion at O2, with enhanced local repressor concentra-
tion through DNA looping. Because aspects of this
concept have previously been challenged (19–21), we con-
sidered other possible scenarios involving RNA polymer-
ase (Figure5).Under inducing
polymerase (red circle) initiates transcription by binding
the Lac UV5 promoter flanked by unoccupied operators
(Figure 5A) or when the unoccupied operators are both
downstream (Figure 5B). Possible outcomes for RNA
polymerase under repressing conditions are shown in
Figure 5C–H. The conventional model is that RNA poly-
merase is occluded in both cases (Figure 5C and F), but
RNA polymerase might be trapped at the promoter as
suggested (19,20) (Figure 5D and G) or might engage
the promoter and dispLace bound repressor (Figure 5E
and H). We designed chromatin immunoprecipitation
(ChIP) experiments to distinguish these models.
Antibodies against the a or s70subunits of E. coli RNA
polymerase holoenzyme were used to immunoprecipitate
promoter DNA fragments that had been cross-linked by
formaldehyde in vivo. A combination of mechanical
shearing and micrococcal nuclease treatment was used to
reduce DNA fragment size to ?200–400bp before reversal
of cross-linking and quantitative PCR with promoter-
specific primers. Signals were normalized to total input
DNA. Results are shown in Figure 6 and Supplementary
Figure S5. Three constructs Lacking E. coli promoters
(BL1076, BL1095 and BL1093) served as negative
controls for RNA polymerase occupancy in the absence
or presence of IPTG. The low background ChIP signal
from BL1093 was set to 1.0 (Figure 6). Enrichment of a
or s70subunits of E. coli RNA polymerase was then
E. coli promoters under repressed or induced conditions.
In all cases, in vivo promoter occupancy by RNA poly-
merase corresponded well to b-gaLactoside reporter ex-
pression. On induction, all constructs with E. coli
promoters showed RNA polymerase
20-foldhigher than background
promoter constructs with a single weak O2 operator,
leaky repression is only 2–3-fold (15,16), and this was con-
firmed for RNA polymerase occupancy (Figure 6, BL546).
For constructs with upstream auxiliary operators without
(BL600) or with (BL596) twisting strain in the repression
loop, RNA polymerase occupancy (as measured by
promoter cross-linking of a or s70subunits) was similar
and at background levels before induction. Assuming that
cross-linked repressed complexes, this result rules out
RNA polymerase capture at repressed promoters in vivo.
For repression loops formed with auxiliary operators
downstream of the proximal O2 operator, results were
similar (Figure 6). If the downstream loop was stable
(BL1050; operators phased so the looped DNA is not
Nucleic AcidsResearch, 2013, Vol.41,No. 1 161
by guest on February 12, 2013
twisted), repression was tight and RNA polymerase was
excluded under repressed conditions (Figure 6). For
downstream loops that are unstable because of dephased
operators, and the requirement for DNA twisting strain
(BL1046) repression is leaky and RNA polymerase holo-
enzyme occupancy is relatively high even under repressing
conditions (Figure 6). Together these results favor the re-
pression models shown in Figure 5C and F. RNA poly-
merase is prevented from binding to the Lac UV5
promoter to the extent that Lac repressor is bound at
the proximal O2operator. As expected, there is general
correlation between IPTG-induced gene expression and
RNA polymerase ChIP signal, and between RR and
RNA polymerase ChIP signal ratio comparing induced
with uninduced conditions (Supplementary Figure S5).
Repression without promoter/operator occlusion
The results described earlier in the text confirm that when
repressor and RNA polymerase compete for overlapping
binding sites, repression is based on the effective local
repressor concentration with contributions by free repres-
sor and repressor looping with similar distance depend-
ence from either upstream or downstream auxiliary
operators. What if repressor and RNA polymerase do
not compete for overlapping binding sites? Using the
single subunit T7 RNA polymerase as a model, it has
been shown in vitro that promoter bending strain, per se,
can be repressive (21). To test this idea in vivo, we created
the constructs shown in Figure 7A and B. When the
auxiliary operator is upstream (Figure 7A), possible T7
RNA polymerase (red triangle) fates under repressing con-
ditions are illustrated in Figure 7C–E. T7 RNA polymer-
ase might be excluded (Figure 7C), captured (Figure 7D)
or it might disrupt repressor and initiate transcription
(Figure 7E). Similar scenarios might occur when the aux-
iliary operator is downstream of the proximal operator
To test these ideas, three constructs were created for
in vivo analysis using T7 RNA polymerase. In one con-
struct (pJ1906; Figure 7A, Supplementary Figure S4), a
T7 RNA polymerase promoter was pLaced between O1
and an upstream Osymoperator, such that the promoter
was approximately two DNA turns from either flanking
operator. Lac repressor binding should not directly influ-
ence T7 RNA polymerase binding at this distance (37).
The operator spacing (86.5bp center-to-center) allows for-
mation of a stable loop. In a second construct (pJ1940;
Figure 7B, Supplementary Figure S4), T7 RNA polymer-
ase promoter and O1positions were preserved, but the
auxiliary Osymoperator was positioned downstream by
58.5bp (center-to-center). As a control, an unlooped con-
struct pLacedthe T7RNA polymerase promoter
Figure 5. Models for possible in vivo behavior of upstream (A) or downstream (B) looping constructs bearing a lac UV5 promoter (black dots
indicate ?35 and ?10 elements) for RNA polymerase (red circle) where the proximal lac O2operator impinges on the promoter. Possible outcomes
under repressing conditions include polymerase exclusion (C and F), polymerase trapping (D and G) or polymerase read-through (E and H).
162Nucleic Acids Research, 2013,Vol. 41,No. 1
by guest on February 12, 2013
approximately two DNA turns upstream of an isolated O1
operator (pJ1938; Supplementary Figure S4).
Constructs were pLaced on the E. coli F’ episome for
testing in the presence of a plasmid encoding arabinose-
inducible T7 RNA polymerase. In vivo testing allowed
determination of b-gaLactosidase expression and the RR.
Data are shown in Figure 8 and Supplementary Table S3.
In the absence of arabinose, T7 RNA polymerase is not
induced, and the reporter signal is low (first two columns
of each set). On T7 RNA polymerase induction, the results
are striking. Even when the T7 RNA polymerase promoter
is well separated from Lac operators, a RR of ?20 is
observed when the promoter is constrained within the re-
pression loop (Figure 8; strain BL1076). In contrast, the
same promoter gives RRs not statistically different from
1.0, when it is pLaced upstream from an isolated O1
operator (Figure 8; strain BL1093) or upstream from a
stable DNA loop formed by Lac repressor binding to O1
and Osym operators (Figure 8; strain BL1095). Slightly
reduced reporter gene expression on full induction of T7
transcription could indicate some favorable interaction
between Lac repressor and T7 RNA polymerase or may
reflect RNA destabilization because of uncoupling of tran-
scription and translation (38).
These results show that DNA looping can repress T7
transcription initiation, but not T7 transcription elong-
ation, and that ‘repression of T7 transcription initiation
can occur in vivo simply by promoter presentation in
tightly bent DNA’. The models shown in Figure 7C and
H are supported by these data. With respect to this mech-
anism, promoter bending deformation may impede T7
RNA polymerase binding, and/or it is possible that T7
RNA polymerase cannot initiate from the tightly bent
promoter because the enzyme Lacks sufficient binding
energy to untwist the constrained DNA within the loop.
It remains to be determined whether promoter pLacement
on the ‘inside’ of the looped DNA (as was the case here) is
important for repression.
CONCLUSIONS AND FUTURE DIRECTIONS
Although perhaps the first and best-studied genetic switch
in biology, several fundamental aspects of Lac promoter
control remain incompletely understood. Using in vivo
studies of simplified constructs that focus on negative
control, this work has sought to clarify four basic prin-
ciples of bacterial promoter repression by DNA looping.
First, is the distance-dependence of DNA loop energetics
similar for auxiliary operators positioned upstream or
downstream from the promoter? Second, what is the
smallest possible DNA loop involving Lac repressor?
Third, is RNA polymerase trapped at repressed pro-
moters? Fourth, when RNA polymerase and Lac repres-
sor do not compete for overlapping binding sites, can a
Figure 6. RNA polymerase and s70occupancy of lac promoters as detected by ChIP and quantitative PCR. Upstream loop constructs are studied
under induced (A) or repressed (B) conditions. Downstream loop constructs are studied under induced (C) or repressed (D) conditions. Black dots
indicate ?35 and ?10 elements. PCR primer sites are indicated by single-headed arrows. ChIP results under conditions of repression (grey bars) or
induction (black bars) are shown for a subunit of RNA polymerase (E) or s70protein (F).
Nucleic AcidsResearch, 2013, Vol.41,No. 1163
by guest on February 12, 2013
promoter be repressed in vivo simply by constraining it in a
strained DNA loop?
Our results suggest that upstream and downstream aux-
iliary operators enhance the concentration of Lac repres-
sor at a promoter-proximal operator with similar distance
dependence. By studying DNA loops not containing pro-
moters, we show that Lac repressor can constrain a stable
loop with as few as four turns of double-helical DNA
in vivo. ChIP results confirm that RNA polymerase exclu-
sion, not RNA polymerase trapping, is the repression
mechanism. Finally, using a T7 RNA polymerase
promoter that does not overlap with Lac operators, we
show in vivo that promoter pLacement within a strained
DNA loop is sufficient to repress transcription initiation,
confirming a previous proposal (21).
Future experiments are planned to extend these results
in two ways. First, it is important to apply ChIP to study
the issue of RNA polymerase exclusion versus trapping in
the context of the wild-type Lac promoter region with an
intact catabolite activator binding protein recognition
sequence under conditions where both positive and
negative control are operative. The spacing between the
?10 promoter element and proximal operator is 6bp in
the wild-type Lac promoter versus 9bp in our experimen-
tal constructs, and it will be important to determine
whether this subtle difference affects RNA polymerase
Figure 7. Models for possible in vivo behavior of upstream (A) or downstream (B) looping constructs bearing a T7 RNA polymerase (red triangle)
promoter (oval) that does not overlap with lac O1or Osymoperators (rectangles). The promoter DNA is curved by looping in (A) but not (B).
Possible outcomes under repressing conditions include polymerase exclusion (C and F), polymerase trapping (D and G) or polymerase read-through
(E and H).
Figure 8. In vivo reporter gene expression from constructs bearing a T7
RNA polymerase promoter (P) two helical turns of DNA upstream of
O1 (BL1093) and with an additional Osym either further upstream
(BL1076) or further downstream (BL1095). Promoter/operator config-
urations are summarized later,
Supplementary Figure S4. In each case, the RR corresponds to the
ratio of the height of the final bar to that of the penultimate bar.
164 Nucleic Acids Research, 2013,Vol. 41,No. 1
by guest on February 12, 2013
fate at the repressed promoter, especially given evidence of
favorable repressor/RNA polymerase interaction (39).
Second, we will study Lac promoter occupancy by archi-
tectural DNA binding proteins to explore the extent to
which the apparent flexibility of the DNA in the Lac re-
pression loop reflects the participation of DNA bending
and kinking by these accessory proteins (15–17,24), a phe-
nomenon that has previously been demonstrated in
looping control at the E. coli gal operon (40–42).
Supplementary Data are available at NAR Online:
Supplementary Tables 1–5, Supplementary Figures 1–5
and Supplementary Methods.
The authors thank Molly Nelson-Holte for technical
Mayo Graduate School, the Mayo Foundation; National
Institutes of Health [GM75965 to L.J.M.]. Funding for
open access charge: NIH.
Conflict of interest statement. None declared.
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