Download Towards evolving a better repressor

Protein Engineering, Design & Selection vol. 22 no. 11 pp. 673–683, 2009
Published online September 2, 2009 doi:10.1093/protein/gzp051
Towards evolving a better repressor
Robert Daber and Mitchell Lewis1
Department of Biochemistry and Biophysics, University of Pennsylvania
School of Medicine, 37th and Hamilton Walk, Philadelphia, PA 191046059, USA
1
To whom correspondence should be addressed.
E-mail: [email protected]
Transcriptional regulation is an essential component of
all metabolic pathways. At the most basic level, a protein
binds to a particular DNA sequence (operator) on the
genome and either positively or negatively alters the level
of transcription. Together, the protein and its operator
form an epigenetic switch that regulates gene expression.
In an effort to produce a ‘better’ switch, we have discovered novel facets of the lac operon that are responsible
for optimal functionality. We have uncovered a relationship between operator binding affinity and inducibility
and demonstrated that the operator DNA is not a passive
component of a genetic switch; it is responsible for establishing binding affinity, specificity as well as translational
efficiency. In addition, an operator’s directionality can
indirectly affect gene expression. Unraveling the basic
properties of this classical epigenetic switch demonstrates
that multiple factors must be optimized in designing a
better switch.
Keywords: induction/regulation/repression/transcription
Introduction
Over 40 years ago, Jacob and Monod (1961a) proposed a
model for transcriptional regulation and introduced the
concept of an epigenetic switch. Subsequently, it was
demonstrated that this switch consists of a protein molecule
(a repressor) and a particular stretch of DNA (an operator)
(Gilbert and Muller-Hill, 1966; Gilbert, 1972). As a unit, the
repressor and its operator are responsible for modulating the
rate at which RNA polymerase can transcribe a gene. Since
its conception, the lac operon has served as a model for
exploring the details of transcriptional regulation. More
recently, the regulatory circuitry of the lac operon has
become a prominent molecular tool used to regulate gene
expression in a number of systems.
One of the most widely used expression systems was
created by coupling the molecular switch of the lac operon
with the more efficient transcriptional machinery of the T7
bacteriophage (Studier and Moffatt, 1986). The pET
expression system is now extensively used for expressing
proteins in bacterial systems. The switch of the lac operon
has also been shown to reversibly regulate gene expression
in non-bacterial organisms (Hu and Davidson, 1987, 1988,
1990; Lee et al., 1997; Caron et al., 2005). Recently, the
switch was used in stem cells to regulate gene expression
during the differentiation process, as well as in mature cells
(Caron et al., 2005). Using an appropriate reporter system,
the kinetics of induction were shown to be ‘rapid, stable and
reversible upon removal of IPTG’, illustrating that the
genetic switch can regulate genes during embryonic development (Caron et al., 2005). Similarly, the lac system can be
used to regulate genes in breast cancer cell lines (Lee et al.,
1997). Some of the most promising work comes from the
successful use of the lac system to regulate gene expression
in mammals, specifically in mice (Mills, 2001). Cronin et al.
(2001) created an ‘optimal inducible system’ where lac provided tight, inducible and reversible regulation of the tyrosinase gene involved in mouse pigmentation. Two lines of
transgenic mice were created: one cell line expressed the lac
repressor and the other expressed the tyrosinase gene under
the control of the lac operator. When the transgenic mice
were crossed, the double transgenic mouse contained a regulated operon.
Although many proteins and enzymes can function outside
of their natural sources, their functionality is generally not
optimized for use in industrial or non-native applications.
The lac repressor, although widely used, is an example of a
molecular tool that has not yet been optimized for its nonnatural applications. The regulated range of expression
obtained with the wild-type switch is well suited for its role
in regulating lactose metabolism in Escherichia coli. In its
natural environment, expression controlled by the switch
cannot be induced unless there is a degree of leakiness
allowing the natural inducer to be made (Jobe and
Bourgeois, 1972). Therefore, the switch permits a basal level
of background expression under repressing conditions to
allow for full activation when needed. Outside of its natural
environment, however, background expression of the regulated genes is not necessary since a gratuitous inducer is
added directly to the system. Since leakiness of the switch
can often be undesirable, we set out to improve upon the
regulatory efficiency of the wild-type switch.
In order to successfully engineer more efficient transcriptional regulators, a complete understanding of each component is required. Therefore, as a first step, we have
undertaken the task of dissecting the interaction between
the repressor and its operator. Of utmost importance in this
interaction is specificity: specificity of the repressor as well
as specificity of the operator. In the absence of discrimination by the repressor for its target DNA sequence, the
repressor protein would wreak havoc in the cell by interfering with alternate regulatory networks. In addition, plasticity in the recognition of repressors by the operator site
would lead to non-specific regulation, and thus inefficient
signaling inside the cell. For organisms to successfully
regulate various cellular processes, each process must
contain regulatory components specific for that pathway. In
addition to specificity, the affinity between the repressor
and its operator is also important. This affinity must be sufficiently high in order for the interaction to be biologically
relevant at the concentrations of repressor and operator
found in the cell. Too weak of an affinity would prevent
association, making regulation impossible. However, the
affinity must be balanced such that the interaction can be
disassembled under conditions requiring transcription of the
regulated gene(s).
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R.Daber and M.Lewis
Early efforts hypothesized that a recognition code existed
with a one to one correspondence between amino acid side
chains and the nucleotide bases (Seeman et al., 1976). Much
effort has been spent on trying to understand specificity of
protein – DNA recognition and binding affinity (Lehming
et al., 1987, 1990; Sartorius et al., 1989). Despite being able
to measure repression among various mutant repressors and
operator sites, little work has focused on understanding the
balance of affinity between the regulatory off and on states in
the lac system. Therefore, designing improved transcriptional
regulators is not possible without further dissection of the lac
operon. The lac operon has withstood years of selective
pressures and therefore, buried in its construction is a wealth
of information on how to design an efficient molecular
switch. Here, we have subjected both the repressor and its
operator to artificial selection in an attempt to further unravel
fundamental properties of a molecular switch that must be
optimized when designing a more efficient switch.
Materials and methods
Creating a library of mutant repressors
A library of mutant repressor molecules was created by modifying the placI plasmid from Novagen. Two silent point
mutations were introduced into the gene, creating a pair of
unique restriction sites (AatII and XmaI) that flank a 76 bp
fragment encoding for the key amino acids on the recognition helix (Besse et al., 1986; Lehming et al., 1987). A
non-functional repressor ( pBD21003) was created on the parental plasmid by introducing three point mutations, Y17P,
Q18A and R22G, which allowed us to select for a gain of
function and minimize the number of false positives in the
selection process. To randomize the critical residues on the
recognition helix, an oligonucleotide was purchase from IDT
with a sequence: 50 -TTA TAC GAC GTC GCA GAG TAT
GCC GGT GTC TCT NNK NNK ACC GTT TCC NNK
GTG GTG AAC CAG GCC AGC CAC GTT TCT GCG
AAA ACC CGG GAA AAA G-30 .
The complementary strand of this oligonucleotide was
generated using a Klenow fill in reaction. In a 30 ml reaction,
1 mg of template DNA was incubated with 33 mmol of
dNTPs (Invitrogen), 5 U DNA Polymerase I (Large Klenow
Fragment New England Biolabs) and 30 pmol of the reverse
primer (50 -CTTTTTCCCGGGTTTTCGCAG-30 IDT). The
double-stranded DNA cassette was purified using a
QIAquick PCR Purification Kit (Qiagen) to remove active
polymerase, salts and unincorporated dNTPs.
The recognition helix cassette was prepared for ligation by
digesting 10 pmol of product with AatII and XmaI. The digest
was then purified using a QIAquick PCR Purification Kit
(Qiagen). The digested cassette was then ligated into the predigested pBD21003 plasmid, and subsequently electroporated
into DH5a cells. A small fraction of the cells (5%) were plated
to establish that there were 1.4 105 transformants. The
remaining cells were used to inoculate a 25 ml cell culture.
This culture was grown to saturation, and the plasmid library
was purified using a QIAprep spin Miniprep Kit (Qiagen).
Creating a reporter plasmid
The reporter plasmid was constructed by placing the green
fluorescent protein (GFPmut3.1) under the control of the lac
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operator or any operator of choice. The reporter plasmid was
created by cloning a region from the pGFPmut3.1
(Clonetech) vector into the pBR322 vector between the ClaI
and EcoR1 restriction sites. The fragment inserted into this
region contained the lac promoter thru the end of the
GFPmut3.1 gene of the pGFPmut3.1 vector. To allow for
facile swapping of operator sites, a Quikchange (Stratagene)
mutagenesis protocol was carried out with oligos (BD002:
50 -TTT CAC ACA GGA AAC AGC TAT GAC CAT G 30
and BD003: 50 TTC CAC ACA ACA TAC GAG CCG GAA
G-30 ) to generate a reporter plasmid without an operator
( pBD1200). From this template, different lac operator variants could be cloned into a reporter by taking the aforementioned primers and adding the sequence of the right half site
to the 50 of the forward primer, and the left half site
sequence to the 50 end of the reverse primer. Each of the
reporter plasmids was transformed into chemically competent
DH5a E.coli cells, and the reporter cells were amplified.
Each of these reporter cells was then made electrically
competent.
Phenotypic screening
Fluorescent-activated cell sorting (FACS) was used to separate bacterial cells into various populations based on the
intensity of their fluorescent phenotype. In short, 110 ng of
the repressor library was transformed into cells containing a
given reporter plasmid. A small aliquot of these cells were
plated to determine the number of total transformants,
whereas the remaining cells were used to inoculate a 10 ml
overnight culture. The following morning, a 25 ml culture
was inoculated 1/100 with the saturated overnight culture.
Once these cultures reached an optical density (OD) (A600)
of 0.6, the samples were transferred to a 48 shaker for
30 min. One OD of cells was then pelleted at 3000 rpm for
5 min at 48C. The cell pellet was resuspended in sterile
5.0 ml of PBS, transferred into a sterile falcon tube and
stored on ice.
Sorting was conducted at the University of Pennsylvania
Flow Cytometry and Cell Sorting Facility. To establish
proper population gates, positive (unrepressed) and negative
(repressed) control samples were analyzed. The library
samples were then sorted into each of these populations, and
the cells were collected in 5 ml falcon tubes containing
1.0 ml of 50% PBS and 50% LB. Cells displaying an intermediate phenotype were also collected. Samples were then
returned to storage on ice until appropriate volumes were
plated on LB plates (150 20 mm). After colonies became
visible on plates, they were chosen to inoculate overnight
cultures for plasmid preparation and sequencing.
In vivo repression assay
To analyze the phenotypes of various repressor– operator
combinations, an in vivo fluorescent assay was used. To
quantitate the level of fluorescence and therefore indirectly
measure the degree of transcription, cells were grown and
analyzed in a Perkin Elmer Victor3 plate reader. In short,
combinations of repressors and operators were transformed
and colonies were selected for overnight culture growth.
Fluorescence was monitored for cultures at all points during
their growth curves and the largest dynamic range between
the repressed and induced samples occurred when cultures
reached stationary phase. Signals at earlier time points made
Evolving a better repressor
it difficult to compare mutants of similar repression strength
because the repressed signal could not be distinguished from
background fluorescence. In general, all assays were conducted by growing each triplicate samples in either 500 ml or
1 ml of LB in a 96-well plate, aliquoting 200 ml samples at
the selected time point then measuring fluorescence (495 nm
excitation wavelength, 510 nm emission wavelength) and OD
(A590). To normalize the signal for each sample, the signal
from the blank sample was subtracted and the resulting fluorescent signals were normalized to cell OD. The normalized
signals from each of the replicates were averaged, and the
standard deviations were calculated (shown by error bars on
plots). In addition, each repression of each repressor and
operator combination was monitored using traditional
B-galactosidase assays.
Reverse transcriptase– quantitative polymerase
chain reaction
To analyze repression more directly, relative mRNA transcript levels were determined using reverse transcriptase –
quantitative polymerase chain reaction (RT– qPCR). Each
repressor – operator combination under question was transformed, and four colonies were selected to inoculate a 5 ml
overnight culture with appropriate antibiotics. The following
morning, 250 ml of each saturated culture was used to inoculate two fresh 25 ml cultures, one containing inducer and the
other without it. At an OD A600 between 0.4 and 0.6, a
500 ml aliquot was removed and processed (Qiagen RNeasy)
to isolate the RNA. Additionally, three 200 ml aliquots were
taken and processed for GFP fluorescence to allow for a
direct comparison of transcript levels to fluorescent signal.
Upon isolation of the RNA, 10 mg of sample was treated
with DNase to remove contaminating DNA (Ambion Turbo
DNA-free Kit). cDNA was then generated (Invitrogen
SuperScript First-Strand Synthesis System for RT– PCR)
using 1.4 mg template mRNA with 75 ng of random hexamer
primers. The resulting cDNA was diluted 1/10 and 1 ml was
used for each triplicate reaction (Stratagene Brilliant SYBR
Green QPCR Master Mix). GFP transcript levels were determined using 300 nM of forward and reverse primers
(BD102F: 50 -CCA TGC CCG AAG GTT ATG TA-30 ,
BD103R: 50 -CGC TTC CAT CTT CAA TGT TGT-30 ). To
compare transcript levels from sample to sample, a housekeeping gene, Ribosomal 16S subunit, was used as a normalizer control (BD112F: 50 -GTG TTG TGA AAT GTT GGG
TTA A-30 , BD113R: 50 -CCG CTG GCA ACA AAG GAT
AA-30 ). All samples were run and processed using thermocycler Stratagene 3000mxP.
Cloning and purification of lac DNA-binding
domain mutants
The wild type and five DNA-binding domain mutants,
Q18M, Y17A, Y17G, Y17R/Q18G and Y17P/Q18A/R22G,
were expressed and purified identically. To allow for
purification using a Nickel column, the dimeric form (residues 1 – 331) of each of these repressors was cloned into the
pET101D vector. The pET101D vector was purchased as
part of a Directional TOPO Cloning Kit (Invitrogen) that
allowed for rapid (5 min) ligation of blunt ended PCR products into the primed vector. To increase the efficiency of
cloning the repressor gene into the vector with the
correct orientation, each repressor gene was PCR amplified
(BD006: 50 -CAC CCA TAT GAA ACC AGT AAC GTT
ATA CGA CG-30 , BD007: 50 -GCC CTC GAG CGC CAG
CGT GGT TTT TCT TTT CAC-30 ) to introduce a directionality tag at the N-terminal region of the gene. Also engineered during the PCR amplification were NdeI and XhoI
restriction sites on the outer edges of the repressor sequences.
The non-cleavable 6-His tag was placed at the C-terminus of
the repressor genes and was shown to not interfere with
protein oligomerization or DNA binding (data not shown).
The expression plasmids were transformed into the chemically competent BL21 DE3 E.coli and plated on LB plates
containing Ampicillin. A colony was selected and grown in a
25 ml cultures at 378C to saturation. The following morning,
a 1-L culture was inoculated 1/100 with the saturated overnight culture. This culture was grown at 378C until an OD
(A600) of 0.4– 0.6 was reached. At this time, the cultures
were induced with IPTG to a final concentration of 2.5 mM.
After 3 h of growth under inducing conditions, the cells were
harvested at 6000 rpm (Sorvall RCB5) for 10 min at 48C.
The pellet was then resuspended in 8 –10 ml of 1 lysis
Buffer (50 mM NaH2PO4, 300 mM NaCl, 0.013 M BME, pH
7.4) and stored at 2808C until needed.
The frozen cells were thawed on ice, and the protease
inhibitor PMSF was added to a concentration of 1.5 mM.
The samples were homogenized in a cell breaker, and the
lysate was then cleared by centrifugation at 15 000 rpm for
30 min at 48C. The resulting supernatant was filtered sterilely
through a 0.45 mM filter and then loaded onto a Nickel
column (His GraviTrap—GE Healthcare) equilibrated with
1 lysis buffer at 48C. The column was subsequently
washed with two column volumes (30 ml) of 1 wash
buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole,
10% glycerol, 0.013 M BME, pH 7.4). To elute the
His-tagged repressor protein, 3 ml of 1 elution buffer
(50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10%
glycerol, 0.013 M BME, pH 7.4) was applied to the column.
The resulting eluate was then dialyzed into 1 l of 1 GF
buffer (200 mM Tris, pH 7.4, 200 mM KCl, 10 mM EDTA
and 3 mM DTT) for 16 h at 48C. After quantifying the dialyzed sample, the protein was aliquoted and frozen at 2808C
until needed.
Electrophoretic mobility shift assay
Differences in electrophoritic mobility were used to determine the relative binding affinities between various
repressor – operator combinations identified in the screening
experiments. The DNA oligonucleotides were designed to
contain clamps outside of the operator sequence to overcome
annealing problems found with short nearly symmetric operator oligos.
Natural operator: BD.GS.100: TTT TCG TAT AAT GTG
TGG AAT TGT GAG CGG ATA ACA ATT CTA GAC
AGG AAA CG, BD.GS.101: TTT TCG TTT CCT GTC
TAG AAT TGT TAT CCG CTC ACA ATT CCA CAC ATT
ATA CG.
Symmetric operator: BD.GS.102: TTT TCG TAT AAT
GTG TGG AAT TGT GAG CGC TCA CAA TTC TAG
ACA GGA AAC G, BD.GS.103: TTT TCG TTT CCT GTC
TAG AAT TGT GAG CGC TCA CAA TTC CAC ACA TTA
TAC G.
Ten picomoles of the top strand were labeled with P32g
ATP using T4 polynucleotide kinase (NEB). The reaction
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R.Daber and M.Lewis
was quenched with 20 ml of Buffer TE and 40 ml of phenol
chloroform isoamyl alcohol. After extracting the aqueous
phase, the probe was cleaned using a centri-spin sizing
column (Princeton separations) equilibrated with TE. The
labeled probe was then annealed with 1.5 M excess of the
complimentary strand and diluted to 220 pM. For each
binding reaction, the repressor protein was serially diluted
and added to a master mix containing 1 binding buffer
(10 mM Tris, pH 8.0, 250 mM KCl, 1 mM EDTA, 5% glycerol, 0.1 mg/ml BSA) and labeled probe to a final concentration of 11 pM. After incubating for 15 min, the samples
were run for 2 h at 200 V at 208C. The gels were subsequently dried at 808 for 45 min and exposed for 16– 20 h
on phosphor storage screens (Molecular Dynamics). The
screens were scanned on a Storm 820 phosphor imager
(Molecular Dynamics), and the images were analyzed with
image quant software (GE Healthcare). Triplicate gels were
run for each repressor– operator combination, and the resulting data were analyzed in origin to determine the dissociation constants.
Results
Plasticity of natural OR1 operator
The components of the switch that regulate the lac operon
have been well characterized (Gilbert, 1972). The primary
operator of the lac operon (OR1), a short stretch of DNA
(17 bp), is composed of two nearly identical half sites and
is located between the end of the LacI gene and the beginning of the lacZ gene (Jacob and Monod, 1961a). The repressor is a 360 amino acid protein that has a modular structure.
It contains an NH2-terminal or ‘headpiece’ domain (60
residues) that binds specifically to operator DNA and a
COOH-terminal ‘core’ domain that binds inducers. The
monomeric repressor self-associates into a dimer of dimers,
where a dimeric repressor molecule recognizes the operator
using the classical helix-turn-helix (HTH) motif. The HTH
provides the scaffold for specific side chains to recognize
bases in the major groove of the operator. Structural studies
(Bell and Lewis, 2000) in conjunction with genetic studies
(Lehming et al., 1988; Sartorius et al., 1989, 1991) have elucidated that the most critical residues for specific operator
recognition are Y17, Q18 and R22 (Fig. 1). These three
amino acids protrude from the recognition helix of the
repressor and form specific interactions with the bases of the
operator. Although previous genetic screens were useful in
demonstrating which residues and operator base pairs are
important for recognition, that work was limited to analysis
of non-natural, symmetric operator sites (Lehming et al.,
1987). Here, we utilize a similar genetic approach but
analyze a larger library of mutant repressors and the biologically relevant, natural operator.
A library of 203 or 8000 mutant repressors was created
by introducing all 20 amino acids into the repressor at the
three critical positions. In the past, transcriptional regulation
has been evaluated by measuring b-galactosidase activity
(Miller units); however, a more direct approach is to measure
the concentration of the reporter protein. A plasmid was constructed such that the expression of a reporter gene,
GFPmut3.1, is regulated by the lac promoter and operator
(Table I). To overcome limitations in screening the repressor
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Fig. 1. The structure of the repressor headpiece bound to the operator. The
recognition helices fit into the major groove of the DNA. On the basis of a
number of crystal structures, we observed three amino acid side chains that
make direct interactions with bases 4, 5 and 6 of each operator half site are
residues Y17, Q18 and R22 (Bell and Lewis, 2000).
Table I. Sequence of operators
Natural OR1
Inverted OR1
Symmetric Left(21)
6
5
4
3
2
1
G
G
G
T
T
T
G
t
G
A
A
A
G
t
G
C
C
C
G
c
–
1
2
3
4
5
6
G
G
G
A
c
c
T
T
T
A
c
c
A
A
A
C
C
C
Sequence alignment of the natural OR1 operator, the ‘ideal’ symmetric left
(21) operator identified by Sadler et al. (1983) and the inverted natural
OR1 operator which results from rotating the natural OR1 operator about the
central G:C base pair.
library on plates, functional mutants were identified by
screening millions of cells and selecting molecules that
block transcription of GFP using FACS. To probe specificity
of the operator to discriminate various repressor molecules,
mutants in the library that repress transcription of GFP were
identified and their genes were isolated and sequenced. Less
than 1% of the molecules in the library are capable of repressing transcription of GFP, illustrating that there are a limited
number of combinations of the amino acid side chains that
are capable of binding to the natural operator and blocking
transcription. This finding is in agreement with previous
analysis which showed that very few changes in the HTH
motif are tolerated (Suckow et al., 1996).
Thirty-three different mutants were identified that block
transcription of the reporter gene and therefore function as
repressors (Table SI, Supplementary data are available at
PEDS online). These altered repressors contain both single
and double site substitutions but no functional repressors
were found with changes at all three sites. Moreover, none of
Evolving a better repressor
the functional mutants had substitutions at position 22
suggesting that the observed interaction between the arginine
and the guanine at position 6 in the operator is critical.
Single site substitutions account for less than half of the
functional mutants; six at position 17 and four at 18. Six of
the single site mutants had previously been characterized as
weak operator binders in Millers herculean analysis of the
repressor (Markiewicz et al., 1994). Although that genetic
analysis produced enormous amounts of valuable data, the
inability to produce all 20 mutants at each residue allowed us
to expand upon the work and identify four novel mutants
also capable of repressing the natural operator as well as
probe the complementarity of mutations at more than one
position. The remaining mutants we identified have substitutions at both positions 17 and 18 with a clear preference
for methionine at position 18. Moreover, the presence of the
Q18M substitution provides additional plasticity; creating
functional repressors that allow 14 different amino acid substitutions at the first position. Since the natural operator contains two distinct half sites and the nature of the interaction
in the second half site remains unclear (Bell and Lewis,
2000; Kalodimos et al., 2002), in the absence of a structure,
it is difficult to determine how the methionine substitution
directly affects operator binding.
These observations are consistent with previous studies
that used the tight binding symmetric operator (Sartorius
et al., 1991); however, it does not appear that the three
mutated positions function independently of one another. We
observed that the first two positions on the recognition helix
function synergistically. For example, Q18L (YLR) does not
bind to the natural operator (Markiewicz et al., 1994) but
functionality is restored by the double mutant Y17M, Q18L
(MLR). Similarly, Y17A is non-functional but the double
mutant Y17A, Q18M (AMR) restores the repressed phenotype. This synergy further supports the notion that there is
not a simple one-to-one correspondence for protein – DNA
interactions and suggests that two neighboring residues may
interact as a single unit on the protein surface. Although we
observe that the 33 mutants are functional, defined as a
2-fold reduction in transcription, and decrease the rate of
GFP production, they do not repress transcription equally.
Repression by all 33 mutants was measured in vivo and
although the mutants are phenotypically similar to the
wild-type repressor, most of the mutants do not repress transcription as effectively (Table SI, Supplementary data are
available at PEDS online). There was, however, an exception: a single site mutant, Q18M, represses better than the
wild-type repressor (Fig. 2A). Traditionally, repression of
Fig. 2. (A) Illustrates the normalized fluorescent signal (see Materials and methods) for samples containing GFP under control of the natural OR1 operator
with the wild-type repressor and Q18M mutants. The third sample contains the ‘ideal’ symmetric left (21) operator repressed by the wild-type repressor. (B)
Decreases in fluorescent signals are due to decreases in mRNA transcript levels of the GFP reporter gene. (C) The binding isotherms are presented for the
purified, dimeric wild-type repressor (filled squares) and the Q18M mutant (open circles) with the natural OR1 operator. The curves demonstrate that the
mutant binds more tightly to the operator than the wild-type repressor. As a negative control (filled triangles), a non-repressing triple mutant, Y18P/Q18A/
R22G, was used. Apparent dissociation constants: wt—26.00 nM, Q18M—10.98 nM.
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R.Daber and M.Lewis
various repressors and operators has been determined by
indirectly measuring the enzymatic activity of the reporter
protein. With current technology, we could more directly
measure repression by comparing transcript levels using
RT– qPCR. Consistent with the observed difference in fluorescent signals, the Q18M mutant reduces the mRNA transcript more efficiently than the wild-type repressor (Fig. 2B).
To verify that increased repression of the Q18M point
mutant was a result of increased binding affinity to the operator and not increased stability of the protein in the cell, its
binding affinity was determined in vitro. Consistent with the
in vivo data, in vitro, the apparent binding affinity of the purified Q18M mutant repressor is 2.5 times greater than that of
the wild-type repressor (Fig. 2C). The consistency between
the data collected in vitro and in vivo suggests that the
increased repression of the Q18M mutant was due to
increased affinity and not increased stability of the mutant
inside the cell. Although the observed affinity is lower than
what has been previously published for the wild-type repressor, the discrepancy could be attributed to the changes in the
repressor resulting in dimerization, the presence of a
C-terminal His tag or buffer system.
Many mutant repressor molecules were identified that bind
to the natural operator more tightly than the wild-type repressor (Schmitz et al., 1978; Schmitz and Galas, 1980;
Swint-Kruse et al., 2003); however, these amino acid substitutions are not directly involved in operator recognition. In
the context of the structure, the Q18M mutant is the only
tight binding repressor that interacts directly with the bases
in the natural operator. Although this mutant was previously
identified (M2) and shown to bind the symmetric operator
with an affinity comparable to the wild-type repressor, its
ability to repress the natural operator was never investigated
(Lehming et al., 1987). Here, we show that altered repressors
that bind specifically to the natural operator and repress
better than the wild-type repressor are rare. From this analysis of 8000 mutated repressors, only one mutant, Q18M,
was identified. Therefore, we would conclude that selective
pressure has established that the wild-type repressor has near
optimal binding affinity to its cognate operator.
Plasticity of the symmetric operator
The operator DNA can be easily overlooked as an active participant with respect to protein –DNA interactions, but
tighter repression can also be achieved by altering the operator. The wild-type repressor binds to a perfectly symmetric
operator more tightly than it does to the natural sequence
(Sadler et al., 1983). This tight-binding operator has a palindromic sequence that corresponds to the left half of the
natural operator with the removal of the central base pair
(Table I). In vivo, this higher affinity interaction translates
into higher levels of repression under non-inducing conditions, essentially reducing the leakiness of the switch. To
help understand what factors may have preserved a lower
affinity operator site, we repeated the artificial selection
experiments with the ‘ideal’ symmetric operator.
Using the repressor library, we selected mutants that
bound to the symmetric operator and repressed transcription
of GFP. In these selection experiments, nearly three times as
many mutant repressors bound to the symmetric sequence
and blocked transcription compared with the natural
operator (Table SII, Supplementary data are available at
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PEDS online). Similar to the mutants who repress the natural
operator, less than one-third of these functional mutants have
single site substitutions. The greatest plasticity occurs again
at the first position (Y17) and includes negatively charged
(E) and positively charged amino acids (K and R). The
second position is less promiscuous, yet 12 amino acid substitutions preserve the ability of the repressor to bind to the
operator, whereas the third position is again invariant. These
results are consistent with previous analysis of the symmetric
operator (Lehming et al., 1990); however, several novel
mutants were also identified (Table SII, Supplementary data
are available at PEDS online).
To validate the differences in repressor recognition
between the two operator sequences, repression of all of the
mutants identified in the selection experiments were
re-analyzed in the presence of both operators. In addition,
several of the mutants were purified and their binding was
analyzed in vitro. Both of these assays verified the selection
results by showing that symmetry in the operator decreases
specificity (Fig. 3A and B). As might be expected, all of the
mutants that repress the natural operator were also found to
repress the symmetric operator. However, contrary to our
expectations, neither the wild-type repressor nor the Q18M
mutant is the tightest binders. Several mutants were found to
bind to the symmetric operator more tightly. In fact, there is
no correlation in repression values when comparing the
binding affinities of mutants to the each of the operators
(Fig. 3C). Analyzing the specificity of repressor recognition
to each of the operator sequences helps to explain why asymmetry in the operator sequence is preferred over a higher affinity interaction. By introducing slight asymmetry between
each operator half site, the switch increases specificity.
Balance of affinity and induction
Changes in both the repressor and operator that lead to a
higher affinity interaction have been identified. Although
higher repression reduces the leakiness of the switch by lowering the basal level of transcription under non-inducing conditions, both the repressor and operator have been preserved
with less than ideal binding affinity. We hypothesized that
increasing affinity between the repressor and operator may
affect the repressor – operator equilibrium and alter the
dynamic range of the switch. The overall effectiveness of the
switch depends upon the amount of transcript produced in
both the off (repressed) and on (induced) states. It is possible
that if the repressor binds to the operator too tightly then the
dynamic range of transcription will be compromised. To
explore the relationship between binding affinity in the
repressed state to the overall effectiveness of the switch, each
of the repressors and operators were analyzed under inducing
conditions.
The dynamic range of the genetic switch was evaluated by
measuring the ratio of GFP produced in the presence and
absence of inducer (Fig. 4A). For all concentrations of IPTG,
the difference in fluorescence between these two states was
greater for the wild-type repressor than the tight binding
Q18M mutant. In both the presence and the absence of
inducer, the mutant repressor binds to the natural operator
more tightly. Similar results were also obtained when repression was determined by measuring the level of the GFP transcript (Fig. 4B). When the fluorescence signal regulated
by the Q18M and the wild-type repressors is plotted as
Evolving a better repressor
Fig. 3. (A) In vivo repression assays with various repressor mutants
demonstrate the differences in specificity between the natural and symmetric
operators. The mutants depicted repress the symmetric operator but cannot
repress transcription more than 2-fold for the natural operator therefore
making them non-functional. Fractional expression defined as GFP signal
with repressor/GFP signal in the absence of repressor for the given operator.
Mutant abbreviations correspond to residues found at positions 17/18/22. (B)
EMSA gel shift images demonstrating that the specificity seen in the in vivo
repression assays is due to specificity in repressor binding. The Y17G
mutant selectively represses the symmetric left (21) operator and shows
minimal affinity for the natural OR1 operator sequence. (C) In vivo GFP
expression assay, there is no correlation between repression strength for
mutants which can repress both the natural OR1 and symmetric operators.
Repression ratios were determined by dividing the normalized GFP signal
for the unrepressed operator sample by the normalized GFP signal measured
for the operator in the presence of the specified mutant repressor.
a function of IPTG concentration, the maximum level of
transcription achieved in the presence of the Q18M mutant
repressor is roughly 2.5 times lower than that observed for
the wild-type repressor. This difference corresponds perfectly
to the measured difference in affinity (2.5-fold) between the
two repressors in the absence of inducer. Essentially, the
mutant repressor binds to the natural operator more tightly in
both the presence and the absence of inducer. The Q18M
mutant binds to the operator and blocks transcription more
efficiently than the wild-type repressor and as a consequence
the conformational change resulting from inducer binding is
insufficient to relieve repression. In an analogous fashion,
the wild-type repressor binds to the symmetric operator in
the presence and the absence of the inducer more tightly
than it binds to the natural operator (Fig. 4A). Again the
tighter binding is only partially relieved by induction, and
the repressor binds to the symmetric operator so tightly that
it cannot be fully induced even at saturating concentrations
of inducer.
Using the thermodynamic scheme shown in Fig. 5 and
assuming that (i) changes in the DNA-binding interface do
not directly impact the residues involved in inducer binding
and (ii) changes at residues 17, 18 and 22 do not alter nonspecific DNA binding, we postulate that all of the mutations
we introduced into either the repressor or the operator
change the equilibrium constant KRO. Modifying the operator
or the DNA binding domain alter the energetics of the
protein – DNA interaction; therefore, changes in KRO are also
manifested in the equilibrium constant KROI. In the context
of expression levels in our in vivo system, all modifications
of the operator or repressor altered the effective [R50]. Since
the changes in this region of the repressor do not affect any
of the other equilibrium constants, the increase in operator
affinity affects repression and induction equally. Therefore,
without also altering the other equilibrium properties which
affect the concentration of active repressor ([Ra]), it is not
possible to produce a tighter repressing switch which maintains full induction. When mutant repressors were previously
identified in genetic screens, the effect of each mutation on
repression was determined, but their effect on inducibility
was ignored (Lehming et al., 1990). Here, the results demonstrate that a delicate balance exists between repression and
induction. In order to design better switches, this balance
must be preserved, and selecting switches which have been
optimized for high repression alone do not produce efficient
transcriptional regulators. An ideal switch must repress transcription under non-inducing conditions as well as derepress
transcription when the gene products are needed. The natural
switch has therefore found an ideal combination of residues
in the recognition helix of the repressor and bases in the
operator to efficiently balance induction and repression.
Role of operator orientation
The natural operator is pseudo symmetric, possessing an
approximate dyad axis about a central G:C base pair.
Mutations in the left half of the operator site appeared to be
more deleterious to repressor binding than the mutations in
the right half of the operator; therefore, it was concluded that
the repressor has a higher affinity for the left half site than it
does for the right (Gilbert, 1972). In E.coli, there are two
additional operators within the lac operon that are recognized
by the repressor. Consistent with the difference in affinity
between the two half sites of the primary operator is the
sequence conservation among the three operator sites. These
three sites contain high conservation in the left half site,
whereas the right half site is poorly conserved. To explore
the role of operator orientation on repressor binding and
repression, the inverted natural operator (OR1) was incorporated into the reporter plasmid, such that the tighter binding
half site was positioned more distal to the promoter
(Fig. 6A). Since operator inversion included the minimal
region of operator needed for binding experiments and crystallization, we anticipated that the orientation of the operator
does not influence repressor binding. To test this hypothesis,
679
R.Daber and M.Lewis
Fig. 4. (A) In vivo induction assays of the wild-type and Q18M mutant repressors with the natural OR1 operator and the wild-type repressor with the
symmetric left (21) operator. Under all concentrations of inducer tested (IPTG), the higher affinity repressor– operator combinations show diminished
induction compared with the wild-type switch. (B) Differences seen in comparing fluorescent signals under saturating inducer concentrations are a result of
lower levels of transcript.
Fig. 5. Equilibrium schematic of the lac repressor. The repressor exists in two different conformations illustrated as R and R*. Mutations in the DNA-binding
domain or changes in the operator result in alterations of the repressor–operator dissociation constant KRO. Since either of these alterations do not directly alter
ligand sensitivity or repressor dimerization, the dissociation constant KROI2 is affected to the same degree. Therefore, increases in repression are also
manifested in diminished induction.
680
Evolving a better repressor
Operator symmetry and translational efficiency
Fig. 6. (A) Sequence alignment of the OR1 and inverted OR1 operator
sequence. The sequence is rotated about the central G:C base pair. (B)
While transcription of GFP under non-repressing conditions is not affected
by the operator orientation, when either the wild-type or Q18M mutant
repressors are present, transcription is more efficiently reduced with the
operator in the natural orientation. (C) Sequence alignment of the
OR2 operator sequence and its inverted partner. (D) Once again, inverting
the operator to place the weaker binding half site distal to the promoter
reduces the level of repression.
we selected mutants from the repressor library capable of
blocking transcription when the operator was inverted. The
same mutants bound to the inverted operator and blocked
transcription of GFP as were observed with the operator in
the correct orientation. However, when these mutants were
analyzed by in vivo repression assays, each mutant failed to
repress transcription of the inverted operator as well as the
natural operator (Fig. 6B). To verify that these results were
not specific to the operator sequence analyzed, the OR2
operator sequence was analyzed in both orientations (Fig. 6C
and D). The results were consistent with both operator
sequences in the presence of each of the mutant repressors.
In addition, the mRNA transcript levels were analyzed, once
again showing consistent results. Since the mutant repressors
cannot discriminate directionality of the operator, differences
in repression must be related to the ability of RNA polymerase to transcribe its message. We suspect that when the stronger binding half site is closer to the start of transcription, the
repressor can more effectively prevent polymerase from transcribing the genes of the operon. In the inverted orientation,
the repressor can be displaced more easily allowing polymerase to transcribe the genes more readily. It therefore appears
that the orientation of an operator is a subtle but important
parameter that must be considered when creating an efficient
molecular switch.
Transcription and translation of a message are usually considered to be independent events; however, we observed that
symmetry in the operator alters translational efficiency. In
the absence of repressor, more GFP is produced when the
promoter region contains the natural operator compared with
the symmetric operator, although the amounts of mRNA produced are nearly identical (Fig. 7). To examine the relationship between operator symmetry and translation of the
message, a series of reporters was constructed that contained
operator regions with DNA sequences that had varying
degrees of symmetry. For each reporter, the fluorescent
signal was measured in the absence of repressor. As shown
in Fig. 7B, as the degree of symmetry increases (as determined by calculating the deltaG of a hairpin formation), the
un-repressed fluorescent signal decreases. This change
demonstrates that there is a clear relationship between the
potential for hairpin formation and the amount of protein
produced. The operator sequence resides at the beginning of
the mRNA transcript, we therefore suspect that symmetry at
the 50 end of the mRNA transcript results in secondary structure formation, which interferes with the translational
machinery. This is consistent with recent findings where
symmetry in codons found in the 50 region of a transcript,
not the operator site, also resulted in decreased expression
levels (Kudla et al., 2009).
It was initially postulated that the repressor could alter the
level of protein expression by blocking either transcription or
translation (Jacob and Monod, 1961b). Subsequent analysis
demonstrated that the repressor regulated the process of transcription exclusively. In this analysis, we have shown that
the operator sequence, not the repressor molecule, is the
component of the switch which can regulate gene expression
at both the transcriptional and translational levels. It is
important to note, however, that the coupling of regulation
for both transcription and translation will only exist for
repressible operators whose binding sites reside after the start
of transcription. In contrast, most activators of transcription
bind to sites upstream of the start of transcription; therefore,
the sequence identity of the binding sites would have no
effect on translation.
Discussion
Transcriptional regulation is complex, as it must achieve the
proper balance of affinity, inducibility, specificity and translational efficiency. Altered operators and repressors exist that
repress transcription more effectively than the naturally
occurring system; however, optimizing repression alone has a
negative effect on the induction and as a consequence the
difference in transcription between the on and off (induced
and repressed) states is less pronounced. Analysis of the
Q18M mutant repressor and symmetric operator suggests that
altering affinity by making changes in either the operator or
repressor DNA-binding domain affects the affinity of the
interaction whether inducer is present or absent. Since the
changes we made in either the operator or the repressor recognition helix occur in a domain distinct from the inducer
binding domain, we can assume that the alterations do not
directly affect the energy of association between inducer and
repressor (KIR). Therefore, this binding constant is nearly
681
R.Daber and M.Lewis
Fig. 7. (A) Transcription of GFP and its mRNA are similar when the natural operator sequence is included in the mRNA transcript. When the operator
contains the symmetric sequence, the level of transcript does not change compared with the natural sequence but the amount of GFP produced is significantly
less. (Signals normalized to signals produced by the natural operator repressed by the wild-type repressor.) (B and C) The unrepressed GFP signal was
measured having inserted operator-like sequences that have different amounts of symmetry. There is a striking correlation between the symmetry of the
operator-like sequence and the amount of GFP produced, suggesting the symmetry at the 50 end of the transcript influences translation efficiency.
identical when the inducer binds to the apo-repressor (RI) or
to the repressor –operator complex (ROI), and any alterations
in the association constant (KRO) between the apo-repressor
and operator must also affect the association between the
operator and the repressor inducer complex (KRIO). Thus,
optimizing repression alone, without also altering other
linked equilibria, results in a switch with reduced inducibility. In other attempts to evolve the lac repressor, the same
balance between inducibility and repression was exemplified.
By randomly mutating the repressor and selecting for
mutants with enhanced sensitivity to inducer, both Lakshmi
et al. and Swint-Kruse et Al. were able to identify mutants
resulting in increased inducibility. A close examination of
682
the background expression by each of these mutants,
however, demonstrates that the increased induction resulted
at a decrease in repression (Swint-Kruse et al., 2003;
Satya Lakshmi and Rao, 2009). To overcome the loss in
repression strength, additional mutations known to increase
DNA-binding affinity were incorporated to produce a mutant
with the proper balance of induced and repressed expression
levels (Swint-Kruse et al., 2003).
In addition to balancing repression and inducibility, an
efficient switch must also balance specificity. In the lac
operon, we suspect that the operator is primarily responsible
for dictating the specificity of the switch. Accordingly, specificity is achieved by introducing asymmetry between each of
Evolving a better repressor
the operator half sites since there are many more mutants
that can recognize a perfectly symmetric operator than one
that is pseudo symmetric. Nearly all operons in the Lac/Gal
family possess pseudo symmetric operators (Weickert and
Adhya, 1992), and we hypothesize that the specificity of
each is inherent in the nature of the operator asymmetry. All
repressor molecules that recognize palindromic operators are
oligomeric and bind to the DNA as a dimer with each
monomer recognizing a half site of the operator. Since symmetric operators contain two identical half sites, the repressor
molecule needs only to recognize that single site, making
symmetric operators less discriminating. When the symmetry
is broken, the repressor must be able to recognize two different half site sequences simultaneously. Thus, the specificity
of the switch is limited by how divergent the operator half
sites can become while still being repressed by a homodimeric repressor containing identical DNA-binding domains.
The degree of asymmetry in the operator sequence also
affects the translational efficiency of the gene(s) regulated by
the switch since the operator resides at the 50 end of the
mRNA transcript. As the degree of symmetry is reduced,
translational efficiency of the message increases, suggesting
that pseudo symmetric operator sites are more ideal. This
demonstrates that the operator portion of the genetic switch
is capable of regulating both transcription and translation in
the lac operon. To avoid complications of regulating translation, operator sequences with non-symmetric half sites are
preferred. This may be another explanation for the conserved
pseudo-symmetry displayed in the operators found in the
LacI/GalR family of operons. To add to the complexity, we
also observed that the orientation of pseudo-symmetric operator alters the level of transcript. Although asymmetry is an
essential parameter for a switch to function efficiently, the
orientation of the operator also needs to be optimized if the
asymmetry generates an operator containing half sites of
different repressor affinities. In the lac operon, higher repression is achieved when the stronger binding site is proximal
to the promoter initiation site.
From the work presented, it is clear that selective pressure
on the lac operon has resulted in a beautifully crafted switch
that has been optimized to achieve a delicate balance of
many factors. Accordingly, it is not possible to reduce the
leakiness of the switch while maintaining full inducibility by
altering the protein – DNA interaction alone. To create a
better switch also requires changing either the inducer
binding site or finding an inducer that can appropriately
relieve the increased repression.
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Received June 10, 2009; revised July 27, 2009;
accepted July 27, 2009
Edited by Jacques Fastrez
Funding
This work was supported by the National Institutes of Health
(GM-44617).
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