A Tale of Two Repressors

doi:10.1016/j.jmb.2011.02.023 J. Mol. Biol. (2011) 409, 14–27

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj ourna l homepage: ht tp : / /ees .e lsev ie r.com. jmb

REVIEW

A Tale of Two Repressors

Mitchell Lewis

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine,37th and Hamilton Walk, Philadelphia, PA 19104, USA

Received 19 January 2011;accepted 9 February 2011Available online8 March 2011

Edited by M. Yaniv

Keywords:operon;repressors;allostery;cooperativity;gene regulation

E-mail address: [email protected] used: NTD, N-te

carboxy-terminal domain; HTH, helcatabolite activator protein.

0022-2836/$ - see front matter © 2011 E

Few proteins have had such a strong impact on a field, as the lac repressorand λ repressor have had in Molecular Biology in bacteria. The genesrequired for lactose utilization are negatively regulated; the lac repressorbinds to an upstream operator blocking the transcription of the enzymesnecessary for lactose utilization. A similar switch regulates the virus lifecycle; λ repressor binds to an operator site and blocks transcription of thephage genes necessary for lytic development. It is now 50 years since Jacoband Monod first proposed a model for gene regulation, which survivesessentially unchanged in contemporary textbooks. Jacob, F. & Monod, J.(1961). Genetic regulatory mechanisms in the synthesis of proteins. J. Mol.Biol. 3, 318–356. This model provides a cogent depiction of how a set ofgenes can be coordinately transcribed in response to environmentalconditions and regulates metabolic events in the cell. A historicalperspective that illustrates the role these two repressor molecules playedand their contribution to our understanding of gene regulation is presented.

© 2011 Elsevier Ltd. All rights reserved.

Introduction

The discovery of the operon marked a pivotalpoint in science—the discovery not only provided amodel for gene regulation but also established a newera in science with the emergence of molecularbiology. By the 1940's, it had been established thatbacteria metabolize sugars for energy production.Most bacteria are versatile and canmeet their energydemands by oxidizing a variety of sugars. Glucose,however, is the preferred sugar source, and it is usedalmost exclusively as an energy source even if othersugars are present. Only when the glucose suppliesare exhausted will alternate sugars, such as lactose,be taken up and oxidized. This ability of the bacteriato switch from one metabolite to another wasdescribed by Jacques Monod as diauxic growth,2 a

penn.edu.rminal domain; CTD,ix–turn–helix; CAP,

lsevier Ltd. All rights reserve

pattern observed when metabolites are used se-quentially rather than simultaneously. Seeking tounderstand the molecular basis of this phenomenonprovides the backdrop for what became the para-digm for gene regulation.Monod developed his ideas about gene regulation

in response to a prevailing hypothesis of the day thattried to describe diauxic growth as a consequence of“enzymatic adaptation”, that is, the conversion of apreexisting enzyme from a nonfunctional to afunctional form in response to particular metabo-lites. The ability of the enzyme to adapt wasbelieved to benefit the organism; thus, theseenzymes were called “adaptive”. Fascinated by theconcept of diauxic growth, Leo Szilard hypothesizedthat constitutive enzyme synthesis was not neces-sarily stimulated by the presence of some inducer,but could also be due to the absence of product,which he described as a classic case of feedbackcontrol.3 As a consequence, Szilard, Monod, andothers abandoned the term “enzyme adaptation” infavor of “enzyme induction”. This promptedMonod to identify and characterize the enzymethat splits the disaccharide lactose into galactose and

d.

http://dx.doi.org/10.1016/j.jmb.2011.02.023

mailto:[email protected]

15Review: A Tale of Two Repressors

glucose. By studying the kinetics of this enzyme'sinduction, Monod found that the addition of ametabolite to Escherichia coli cells actually increasedthe production of the enzyme, now called β-galactosidase.2,4 Over the next several years,Monod explored the biochemistry and genetics oflactose metabolism. He determined that the naturalinducer of β-galactosidase is allolactose, an analogof lactose that is created by a side reaction of thisenzyme. Several effector molecules that mimic thenatural inducer and affect the synthesis of β-galactosidase were synthesized. A particularlyeffective gratuitous inducer is isopropyl-β-D-thioga-lactopyranoside (IPTG). Monod also identifiedbacterial strains that altered the synthesis of β-galactosidase. Many altered strains abolished theability of the bacteria to make an active enzyme,while others changed the inducible character ofenzyme synthesis by constitutively producing β-galactosidase, that is, the de novo synthesis of theenzyme proceeded in the absence of an inducer. Thefundamental questions that confronted Monod wereas follows: what is responsible for inducible andconstitutive enzyme synthesis; how do inducersincrease the production of β-galactosidase; and howdoes this relate to diauxie?While Monod was focusing on lactose metabolism

in one corridor of the Pasteur Institute, Andre Lwoffwas exploring the mechanism of phage develop-ment down the hall. Lwoff noted that, in someinstances, a virus can infect its host and replicate,producing hundreds of viral progeny. While at othertimes, the virus takes up residence in the host,presumably integrating its DNA into the hostchromosome, establishing a prophage. Under nor-mal conditions, these lysogenic phage are highlystable and replicate passively along with thebacteria. However, in response to specific environ-mental stimuli or stress, the virus can switch fromlysogenic to lytic development in a process referredto as prophage induction.5 Lwoff wanted todetermine how phage can live quiescently with itshost and then reappear when confronted withenvironmental change. Although it was not readilyapparent at the time, the fundamental questionbeing asked by Lwoff was in fact similar to thatbeing asked by Monod: how are cellular eventsturned on or off?Francois Jacob entered Lwoff's laboratory to work

on phage development and the mechanism oflysogeny. Jacob with Elie Wollman exploited therecently discovered phenomenon of bacterial conju-gation to explore the mechanism of prophageinduction.6 When a chromosome bearing a λprophage enters a nonlysogenic recipient cell, halfof the bacteria lyse and produced phage particles. Incontrast, when a nonlysogenic donor is crossed witha lysogenic recipient, none of the bacteria releaseviral progeny. Jacob and Wollman concluded that

the immunity of the lysogenic bacteria is caused by acytoplasmic factor.7

Bacterial conjugation also proved useful forexploring bacterial functions and the genetics oflactose metabolism. Having isolated a number ofmutants that affected the production of β-galactosi-dase, Monod reckoned that mutants could becrossed in various combinations and that therelationship between the inducible and constitutivenatures of enzyme synthesis could be determined.Published in 1959, the PaJaMa experiments [namedfor Pardee, Jacob, andM(a)onod] demonstrated thatinducers function by relieving inhibition, consistentwith the hypothesis of Szilard.8 Moreover, thereappeared to be molecules in the cytosol that areresponsible for regulating the expression of thestructural genes. As a consequence, the formerlyaccepted “induction model” of enzyme synthesiswas cast aside in favor of a “repression model”.Prophage induction and galactosidase production

are, at first glance, two unrelated phenomena, yetboth processes could be controlled in fundamentallysimilar ways. Jacob recognized that the synthesis ofthe genes of the lac system and the synthesis of genesrequired for lytic development might be regulatedby a common mechanism. In both systems, amolecule in the cell appears to block gene expres-sion, and this repression could be reversed by asignal, either a metabolite in the case of lac or UVirradiation in the case of λ. These cytosolic mole-cules were called “repressors”.In 1961, Journal of Molecular Biology published

Jacob and Monod's manuscript entitled “GeneticRegulatory Mechanisms in the Synthesis of Pro-teins” that outlined how a set of genes might becoordinately regulated, providing a model for generegulation.1 Jacob and Monod described an operonas a simple control circuit where a collection ofstructural genes could be regulated in a coordinatedfashion. These structural genes code for a group ofproteins that are responsible for a particularmetabolic function. Regulating the ensemble ofproteins requires a master switch, called therepressor molecule, which is produced from aregulator gene (lacI coding for the lac repressorand the cI gene coding for the λ repressor). When theregulator binds to a specific site on the genome, anoperator, it blocks transcription of the structuralgenes. The operator, a small fragment of DNA, wasoriginally identified by Monod as cis-acting consti-tutive (oc) mutants. These mutants were locatedbetween the end of the gene that codes for therepressor (lacI) and the beginning of the structuralgenes lacZ, lacY, and lacA, which code for threeproteins involved in lactose metabolism: β-galacto-sidase, lac permease, and a transacetylase. Repres-sion is relieved when a regulatory protein binds ametabolite or an inducer, which lowers or eliminatesthe affinity of the repressor for the operator (Fig. 1).

repressor

lacZlacI

RNA polymerase

operatorpromoter

lacZlacI

RNA polymerase

operatorpromoter

inducer

Fig. 1. Diagram of the operon model of Jacob and Monod. The repressor molecule that was produced by the lacI genebinds to operator site and blocks transcription of a set of structural genes (lacZ) that follows the operator site. Therepressor when bound to an inducer (marked by a yellow star) decreases the affinity of the repressor for operator andallows transcription of the set of structural genes.

16 Review: A Tale of Two Repressors

A similar model could be used to explain the lifecycle of the phage. A repressor molecule, producedfrom a cI regulator gene, binds to an operator siteand blocks transcription of the phage genes neces-sary for lytic development. In this instance, repres-sion is relieved by an environmental stimulus, suchas UV irradiation rather than a metabolite. Themodel for gene regulation proposed by Jacob andMonod was met with criticism, for it was equallyplausible that the whole operator is transcribed aspart of the mRNA and that the repressor wouldregulate translation of the message into protein.Moreover, the molecular composition of the repres-sor molecule was unknown: it could be eitherprotein or nucleic acid. To get to the answer, theregulatory gene products had to be isolated, andtheir mode of action had to be established, butisolating these repressors would not be a simple feat.

Isolation and Characterization of theRepressors and their Operators

The number of repressor molecules in the bacterialcell is small and constitutes a minor fraction of thetotal cellular protein, thusmaking it difficult to purify.Nevertheless, both lac and λ repressors were purifiedin the mid sixties, albeit using very differentapproaches. Benno Muller-Hill, working with WalterGilbert, identified a potential lac repressor protein by

isolating cell fractions that bound a radioactivelylabeled inducer.9 Since there were no known inducermolecules of λ repressor, Mark Ptashne developedan alternate strategy.10 He differentially labeled twocultures of bacteria, one infected with a λ strain thatproduced a functional repressor and anotherinfected with a λ strain that was incapable ofmaking repressor. The cell cultures were mixedtogether, sonicated, and the soluble extract wasfractionated. Only one fraction appeared that wassingly labeled and presumably contained the repressormolecule.Once these proteins were isolated, the critical

experiments could be performed. Ptashne demon-strated that the selectively labeled protein fractioncontained a molecule that co-sediments with thewild-type λ operator DNA but not with DNA thatcontains a defective operator, a λ virulentmutation.11 Similarly, Gilbert and Muller-Hill ob-served that the isolated fraction, which contained amolecule that binds to an inducer, co-sedimentswith the lac operator DNA but with the DNAcontaining an operator constitutive mutation. More-over, the addition of a gratuitous inducer preventedthis molecule from sedimenting with the operatorDNA.12 Gilbert and Ptashne had isolated the tworepressors molecules, demonstrating that at leastpart of the operon model proposed by Jacob andMonod was indeed correct. Attention now focusedon exploring the finer details of gene regulation.


The first step was to study the biochemicalproperties of these proteins. However, the amountof protein initially recoveredwas too small for furthercharacterization. Using a variety of clever geneticmanipulations, Muller-Hill et al. created alteredbacterial strains that produced large amounts of lacrepressor.13 The over expressed protein was purifiedand used initially for amino acid sequencing14,15 andstructural characterization.16 The lac repressor con-tains 360 amino acids that self-associate, forming atetrameric assembly. Each repressor monomer has amodular structure; when treated with trypsin, it iscleaved into twodomains: a smallN-terminal domain(NTD), which was referred to as the headpiece, and alarger C-terminal fragment, called the core. Theheadpiece recognizes the operator, and the corebinds to effect or ligands.16 Once the gene for λrepressor was cloned and overexpressed, the repres-sor protein could also be purified and characterized.At 236 amino acids, λ repressor is smaller than lacrepressor, but it also self-associates, forming higher-order oligomers. Each λ repressor monomer iscomposedof twodomains; similar to the lac repressor,these domains are tethered by a protease-sensitivelinker.17 The NTD binds to DNA, and the carboxy-terminal domains (CTDs) allow the λ repressor toself-associate.Attention focused on characterizing not only the

repressor but also their site of action, the operator.cis-acting constitutive (oc) mutations were originallyused to map the location of the lac operator betweenthe end of the lacI gene and the beginning of the lacZgene.18 Roughly 10 years later, the operator site wasisolated,19 allowingGilbert andMaxam20 to sequencea 27-base-pair section of the double-stranded DNA.The lac operator sequence is pseudo-symmetric,possessing an approximate dyad axis about a centralG·C base pair. The minimal size of operator requiredfor specific bindingwas later shown to be only 17 basepairs and corresponds to the center of the originallyfound 27-base-pair sequence.21 The regulatory regionon the phage genome is considerably more complex;two operator regions exist, OR and OL, that areseparated by ∼2.4 kb.22 Once the operators wereisolated and sequenced, Ptashne established that ORand OL are both roughly 85 base pairs and containthreeDNAbinding sites that occur in tandem. TheORregion consists of operator sites OR1, OR2, and OR3,with a similar arrangement of three operators for OL.All six operators are ∼17 base pairs and containsimilar but not identical sequences with pseudotwo-fold symmetry.23

How do these repressors recognize a specificoperator sequence of DNA? Seeman et al. suggestedthat sequence-specific DNA recognition could beachieved by having the amino acid side chainshydrogen bond to the exposed bases in themajor andminor grooves of the DNA.24 Unfortunately, despiteconsiderable effort, the three-dimensional architec-

ture of these molecules remained elusive for manyyears. As a consequence, a fallback position wastaken, and attention was focused on the structures ofthe isolated DNA binding domains. The NTD of λrepressor adopts a globular structure that containsfive α-helices.25 A bi-helical unit, now referred to as a“helix–turn–helix” motif (HTH), protrudes from thesurface of the molecule. In the crystals, the NTD self-associates, albeit weakly, forming a dimer. As aconsequence, two HTH motifs, one from eachmonomer, are oriented to potentially fit into succes-sive grooves of the operator. Later, the crystalstructure of the NTD bound to a synthetic operatorverified that the NTD of λ repressor uses the HTHmotif to recognize a 17-base-pair nucleotide contain-ing an operator sequence.26,27 The 2-fold axis of theprotein dimer is aligned with the pseudo-2-fold axisof the operator. Amino acid side chains on the HTHmotif recognize the operator by forming hydrogenbonds to the bases in themajor grove of each half site,and recognition is achieved by a handful of specificinteractions. The headpiece domain of lac repressorbound to DNA was determined in solution. It is asmall compact, globular structure that also containsan HTH motif.28 As was seen with λ repressor, theside chains that protrude from the HTH motif makesequence-specific interactions with the two half sitesof the operator. In addition, hydrophobic interac-tions contribute to the specificity and binding affinityof the complex. As it turned out, this HTH motif is acharacteristic of many DNA binding proteins.29

After seeing how the repressor recognizes itsoperator, attention turn to the CTD of the repressorsand to the regulation of thesemolecular switches. Thecore domain of the lac repressor is composed of twosubdomains that each contain a six-stranded parallelβ-sheet sandwiched between four α-helices.30 Thequaternary structure of the repressor is unique; themolecule self-associates into an unusual tetramer thatappears roughly as a V-shaped dimer of dimers. TheCTD of λ repressor also tends to self-associate,forming dimers, as well as tetramers and octamers.31

A monomeric unit folds into a highly twisted seven-stranded antiparallel β-sheet followed by a singleturn of the 310-helix.

32

The structures of these domains provided onlyfragmental information as to how these repressorsfunction. It was therefore imperative to determine thearchitecture of the intact molecules, as well as thesemolecules bound to various ligands, effectors, andoperators. Eventually, the structure of the intact lacrepressor was solved both in the presence and in theabsence of its operator.33 The protein scaffold of theintact lac repressor contains four discrete functionalunits: the headpiece, a hinge region, a sugar bindingdomain, and a C-terminal helix33 (Fig. 2). Theheadpiece, which binds to the DNA, is connected tothe core by a stretch of polypeptide chain called thehinge region. In the absence of DNA, the hinge region

Fig. 2. A ribbon diagram of the quaternary structure of the lac repressor complexed to DNA. Lac repressor is a tetramericstructure where each monomer is drawn in color. Each dimeric repressor binds to a 21-base-pair duplex deoxyoligonucleo-tides. The observed lac tetramer appears to be a tethered dimer of dimers. Below is a view of the lac repressormonomer fromthe complex with DNA. Four functional domains occur in repressor monomer: starting from the N-terminus (1), the DNAbinding domain or headpiece (residues 1–45) is colored red; (2) the hinge region (residues 46–62) is colored yellow; (3) theligand binding domain or core (residues 63–329) that has distinctN- andC-terminal subdomains are colored in two shades ofblue; and (4) the tetramerization helix (residues 340–357) is colored purple. The monomers associate into a functional dimerthat binds to the operator. Dimer is shown bound to the anti-inducer orthonitrophenylfucoside.


is disordered, and the headpiece domain is highlymobile. In contrast, when the headpiece is bound tothe operator, the hinge region adopts a helicalconformation that also makes sequence-specific inter-actions with the operator and recognizes bases in theminor groove. The hinge helices pry open the minorgrove anddramatically bend theDNA,deforming thecenter of the operator from a canonical B formconformation. The significance of this deformationand bending, however, remains unclear. The core ofthe repressor consists of two structural domains; the

C-terminal subdomain is responsible for dimeriza-tion, while the N-terminal subdomain performs theimportant role of communicating to the headpiecewhether a metabolite is present. The lac repressorterminates with an α-helix that self-associates, form-ing a four-helix bundle to create the biologicallyobserved tetrameric repressor.The full-length repressor λ repressor bound to its

operator DNA confirmed that the two distinctdomains of known structure are tethered by ashort segment of polypeptide (Fig. 3).34 Although

(b)

NTDNTD

CSR

CSR

CTD

CTDCTD

CTD

NTDNTD

(a)

Fig. 3. Orthogonal views of the λ repressor–operator complex. One subunit of the dimer (salmon) adopts an extendedconformation, and the other adopts a compact conformation (blue). The radii of the spheres were determined on the basisof the actual size of the NTD and CTD. The NTD and CTD are connected by a pair of antiparallel β-strands, and aconnecting loop that contains the site of RecA-mediated autocleavage is highlighted in red. The DNA is shown in pink.The symmetry axes of the individual domains are shown as green rods and are nearly perpendicular to one another.


this linker was previously assumed to be flexibleand void of structure, it is an integral part of theCTD, forming a pair of antiparallel β-strands thatdrape across the surface of this domain. The mostremarkable aspect of the intact dimeric structure isits lack of global symmetry. The dimeric repressordoes not have a single axis of symmetry; the 2-foldaxes that relate the NTD and CTD dimers aresubstantially skewed and are nearly orthogonal toone another.

λ-Cooperativity and Gene Regulationfrom a Structural Perspective

Regulation of the virus life cycle requires a controlmechanism that is more elaborate than a simple bi-stable switch. At the heart of the system is therepressor protein, which unexpectedly functions asboth a repressor and an activator of transcription.

When λ repressor binds to its operators, it not onlyblocks transcription of the viral genes necessary forlytic growth but also stimulates transcription of itsown gene. Figure 4 is a schematic representation ofthe right operator region, illustrating the binding ofthe λ repressor to operators in a λ lysogen.When λ repressor binds to adjacent sites on the

DNA, the proteins interact cooperatively. Coopera-tivity is a consequence of a linked equilibrium,which manifests itself by a left shift of a bindingisotherm. The affinity of the repressor for OR1 isgreater than that for OR2 or OR3, as a consequencecooperativity facilitates or increases the bindingaffinity of a second dimer to the lower-affinity siteOR2.35 Binding to OR2 increases the level ofrepression of PR. The physiological consequence ofthe cooperative interaction is to decrease the amountof repressor needed to maintain a lysogen.36

Cooperativity is not only essential for lysogeny; italso amplifies the sensitivity of the switch that

OR1OR3 OR2

PRM PR

C

N

C

N

C

N

C

N

cooperative interaction

Fig. 4. The interactions of λ repressor at the rightoperator regions in a λ lysogen. The repressor dimersbound cooperatively to adjacent operator sites in OR andOL. The repressor monomer is shown in blue and cyan..Each subunit of the dimer consists of an N-terminal DNAbinding domain (N), a C-terminal oligomerization domain(C), and a linker region (black) connecting the two. Thedimer pair bound cooperatively at OR1 and OR2 repressestranscription from PR, and the dimer bound at OR2 alsoactivates transcription from PRM. Dimer pair bound alsobinds cooperatively at OL1 and OL2 represses transcrip-tion from PL (not shown).


controls prophage induction.37 Mutations that dis-rupt cooperativity, while having no effect on thebinding of a dimer to a single operator site, werefound within the CTD and lie near the interfaceformed when two repressor dimers associate into atetramer.38,39 A structural model of cooperativity(Fig. 5) was created by superimposing the intactdimer onto the structure of the CTD tetramer.34 Themodel illustrates that when a cooperativity complexis established, the repressor dimers bind to thelateral sides of the operator while the CTDs arepositioned medially.The repressor concentration ultimately dictates

whether the wild-type phage will be maintained as alysogen or enters lytic growth. Binding of a repressorat OR2 not only increases repression of PR but alsostimulates transcription of the cI gene, which codesfor the λ repressor, by increasing the affinity of RNApolymerase for the promoter PRM.

40 Mutant repres-sors that bind to the DNA normally, but that fail toactivate transcription, have amino acid substitutionon the HTH motif albeit distinct from amino acidsrequired for operator binding.41 By activating thetranscription of its own gene, the phage ensures thatthere is sufficient repressor for maintaining thelysogen. Of course, an excessive amount of repressorwould impede the ability of the phage to enter lyticgrowth. Consequently, the repressor maintains asteady-state level of its own gene product by bindingto the operator OR3 and by blocking the transcriptionfrom PRM. This autoregulation requires communica-tion between OR and OL.

42

Cooperativity at a distance requires communica-tion between the left and right operators. Duringlysogenic growth, binding of λ repressor to OR1 and

OR2 and to OL1 and OL2 represses transcription ofthe early lytic genes by physically blocking theaccess of the polymerase to the promoter. Acooperatively bound pair of dimers at OR1 andOR2 can interact with a cooperatively bound pair ofdimers at OL1 and OL2 by forming a higher-orderedoctameric complex. When this octamer forms, theintervening 2.4 kb of DNA form a loop thatjuxtaposes OL3 and OR3. The close proximity ofOL3 and OR3 promotes a pair of dimers to bindcooperatively to these two operators. As a conse-quence, OR3 is occupied at lower concentrations ofrepressor than would be predicted simply by itsintrinsic affinity for this operator.42 Mutation of OL3,in the context of an otherwise wild-type λ phage,results in a substantial defect in prophage inductiondue to loss of negative autoregulation.42 Figure 6a isa model of cooperativity at a distance that wascreated by superimposing the dimeric repressoronto the crystal structure of the CTD octamer. Thisassembly brings the operators OR3 and OL3 intoclose proximity. Figure 6b illustrates how tworepressor dimers can associate and negativelyregulate transcription of PRM. The model wascreated by superimposing the intact dimer ontothe tetrameric CTD.Prophage induction or the entry into lytic growth

occurs with the destruction of the repressor,allowing RNA polymerase access to the promotersPR and PL and transcription of the lytic genes. Therepressor self-destructs by an autocleavagereaction.43 The cleavage hydrolyzes a peptidebond, which physically separates the NTD fromthe CTD.44 The amino acids that perform thiscatalysis are a pair of conserved residues, serineand lysine, that are located within a shallow grooveon the surface of the CTD. The mechanism for theself-cleavage is analogous to the serine proteases;45

the lysine residue, in an unprotonated form,removes a proton from the serine residue, therebyactivating a nucleophile that attacks the carbonylcarbon atom of the peptide bond in the linkerregion.43 Quite unexpectedly, these catalytic aminoacids are located at the cooperativity interface.Consequently, the quaternary structure of therepressor must be disrupted for the active-siteresidues to be exposed and find its substrate. Thecleavage reaction occurs in response to stress. DNAdamage activates the SOS response and the cellularRecA protein.46 How RecA facilitates the cleavagereaction is not well understood. Mutations thatinhibit dimerization have an increased susceptibil-ity to RecA-mediated cleavage, and converselyrepressor that form more stable dimers are resistantto RecA-mediated cleavage. It appears that RecAmay associate with the repressor and may alter thedimer–dimer equilibrium. Mutant repressors thatprevent the RecA-mediated cleavage render thelysogen non-inducible.47

(a) (b)


Lac Allostery and Gene Regulation froma Structural Perspective

At first glance, the regulatory circuit that controlsthe lac operon appears to be a simple biphasic system;binding of lac repressor to its operator physicallydeniesRNApolymerase access to a promoter. In 1963,Monod et al. published in Journal of MolecularBiology a model that describes how metabolitesmight play a pivotal role in gene regulation.48 Thefundamental tenet of their model is that activity of aprotein can be modulated by altering its conforma-tion. Effector molecules (metabolites) when bound toa protein have the potential to alter the structure and,

(a)

(b)

(c)

cooperativity interface

N

N

C

C

C

C

NNN N

C

CC

C

N N

C

C C

C

Fig. 5. Structural modeling depicting the interaction ofadjacently bound λ repressor dimers. (a) The pairwisecooperativity complex was modeled by superimposingthe CTDs of two intact DNA-bound dimers onto thestructure of the CTD tetramer. This superimpositionbrings the DNA of one repressor–operator complex intorough alignment with that of the second complex. The C-terminal α-helices of the NTDs are also shown, whichmediate formation of a weak NTD dimer. (b) The pairwisecooperativity complex is rotated by 90° to illustrate thatthe NTDs bind on opposite sides of the DNA with theCTD tetramer below. (c) The model of the cooperativitycomplex is rotated by 180° with respect to (a) in order toview the cooperativity interface.

Fig. 6. A model depicting the higher-order loopedcomplex that facilitates negative autoregulation. Thecooperatively bound λ repressor dimer pair at OR1 andOR2 interacts with the cooperatively bound repressor atOL1 and OL2, forming an octameric complex that loops2.4 kb of intervening DNA. (a) The model was created bysuperimposing the CTDs of four DNA-bound dimers ontothe structure of the CTD octamer. In this case, thesuperimposition was performed by first constructingtwo pairwise cooperativity complexes. (b) Formation ofthe looped complex juxtaposes OL3 and OR3, allowinganother pair of repressor dimers to bind cooperatively tothese sites. A model depicting the interaction of nonadja-cently bound repressor dimers shows how two repressordimers might look when bound cooperatively to OL3 andOR3 in the context of the looped complex.

consequently, the protein's ability to perform aspecific function at a distal site. Building on thismodel, Monod et al. published a rigorous descriptionfor how a conformational transition in multimericproteins alters the ability of the molecule to perform agiven function.49 Although this theory of allosterictransitions,which became knownas theMWCmodel,did not explicitly mention the lac operon, it would besafe to assume that they anticipated that the lacoperon would be allosterically regulated.The repressor adopts two distinct conformational

states: one bound to the operator and another whenbound to inducer. This was first illustrated byobserving that crystals of the repressor-operatorcomplex when exposed to an allosteric inducerimmediately shattered.50 This is, of course, reminis-cent of the shattering of crystals of deoxyhemoglobinwhen exposed to air.51 The two distinct conforma-tional states of the repressor, R and R* (designated asR and T by MWC), are related by an equilibriumconstant KRR⁎. The linked equilibria that describe thebinding of the repressor to its operator and to theinducer are illustrated in Fig. 7. There are 12 repressorspecies that contribute to the total repressor concen-tration. The amount of repressor, R, that is able to bind

Fig. 7. A diagram illustrating the linked equilibria. KRO is the equilibrium constant that describes the binding of therepressor (R) to the operator (O).The equilibrium constant KRR⁎ is an inherent property of the repressor and dictates theratio of the repressor in the active and inactive conformations. The inducer binds to the repressor with affinities dictatedby KIr and Kir⁎, the binding of the inducer to these two conformational states. Below is a schematic representation of thedimeric repressor in the active and inactive conformations.


to the operator depends upon the conformationalequilibrium, the inducer concentration, and theinducer binding affinities to the R and R* conforma-tions. These three parameters are responsible ofestablishing the relative amount of active and inactiverepressors. For the lac repressor, the conformationalequilibrium constant is close to unity, and in solution,the two conformations are readily interchangeablewith the inactive conformation (R*) being modestlyfavored over the active conformation.52The role of the inducer molecule is to shift the

apparent equilibrium KRR*, driving the repressortoward the inactive or R* conformation. At highconcentrations of inducer, the apparent value of

KRR* increases by several orders of magnitude, and alarger fraction of the total repressor concentrationadopts the R* conformation, incapable of binding tothe operator. Inducers shift the apparent equilibri-um by forming an intricate hydrogen-bondingnetwork that cross-links the NTD and the CTD.Figure 8 illustrates the extensive hydrogen bondingbetween the inducer and the repressor. All of theoxygen atoms of the galactose ring form hydrogenbonds either by directly bonding to the amino acidside chains or mediated through water molecules.Anti-inducers bind to the repressor, but form adifferent hydrogen-bonding network. As seen inFig. 9, the anti-inducer binds to the repressor, in the

S193

S191O6´

R197

D149

N246

S69

N125

Fig. 8. This figure illustrates the binding of the inducer(IPTG) to the repressor. The dark-blue portions of thestructure correspond to residues in the CTD, while thelight blue corresponds to the N-terminal portion of thestructure. The inducer and the water mediate hydrogenbonds that stabilize this conformation of the repressor.


presence and in the absence of operator, by makingalternate interactions. In general, inducers have a C6hydroxyl, which is used to establish a water-mediated hydrogen-bonding network. While thishydroxyl is a necessary prerequisite for induction, itis not sufficient. The functional groups attached atthe C1 position also play a crucial role in discrim-inating inducers from non-inducers and in deter-mining the potency of the inducer. In order for agalactoside containing the C6 hydroxyl to be apotent inducer, it must have a C1 substituent groupthat sufficiently increases the binding energy whilemaintaining conformational flexibility.53

The allosteric signal is transmitted through thedimer interface. Over 4000 single amino acidsubstitutions of the repressor were created, andsome of which resulted in mutant repressor mole-cules, classified by an Is phenotype, that bind tooperator DNA with wild-type affinity but areincapable of induction.54 Over half of these Is pointmutations appear in the effector binding pocket anddirectly alter the affinity of the inducer for therepressor. Other mutations that produce the Is

phenotype cluster at the monomer–monomer inter-face between the N-terminal subdomains (Fig. 10).From the position of these mutations, it appears thata signal is transmitted from the effector site, throughthe dimer interface, to the hinge helices and theDNA binding domains. The inducer stabilizes a

conformation of the repressor that precludes theheadpiece and the hinge helices from efficientlybinding to the operator.How can a biphasic switch produce levels of β-

galactosidase that increase linearly as a function ofinducer concentration? This was an issue that greatlyconcerned Monod.3 The answer was arrived at byJacob after watching one of his sons playing with hiselectric trains. An on-or-off switch can function suchas a rheostat by oscillating between the on and offstates with different frequencies.3 The fraction oftime the promoter is accessible and the duration oftime it is free are both relevant for effectivetranscription. The kinetics of transcriptional initia-tion of the lac operon have been well characterized.55

RNA polymerase binds to the promoter region,forming a “closed” complex, which then undergoesan “isomerization” to establish the “open” (strandseparated) initiating complex, a process that takes∼60 s. In the absence of an inducer, the repressorbinds to the operator and is released in a stochasticfashion. On average, the amount of time therepressor is bound to the operator greatly exceedsthe amount of time it is free. Based on the kineticdata of Riggs et al.,56 the operator and the repressorform a stable complex that has a lifetime of∼1600 s. Of course the complex dissociates, butthe operator is only vacant for ∼6 s before the twore-associate. As a consequence, there is not enoughtime for the polymerase to bind to the promoterand to initiate transcription, and the switch iseffectively turned off.The inducer decreases the affinity of the repressor

for the operator by 3 orders of magnitude, whichcould be a consequence of either decreasing the onrate, increasing the off rate, or a combination of thetwo. If the inducer only altered the dissociation rateof the repressor, then the mean occupancy timewould be significantly reduced; however, the meantime the operator is free of repressor would beunchanged, and therefore, on average, the timeframe would still be too short for transcription toinitiate. In contrast, if the inducer altered theequilibrium by depressing the on rate, then theaverage time the DNA is free of repressor wouldincrease sufficiently, allowing transcription to initi-ate. At high inducer concentrations, the off rate wasestimated to increase ∼10-fold,57 which implies thatthere is a substantial decrease in the on rate. Alteringthe association rate rather than the dissociation rateallows polymerase to initiate transcription andproduce levels of β-galactosidase that increaselinearly as a function of inducer concentration.Cooperativity at a distance may also play a role in

regulating the lac operon. On the bacterial genome,there are two stretches of DNA that resemble the lacoperator; one of the sequences, O2, is 401 base pairsdownstream of the primary operator, and the other,O3, is 92 base pairs upstream of O1. Although these

image of Fig. 8

Fig. 9. The binding of the anti-inducer orthonitrophenylfucoside to the repressor in the absence and in the presence ofthe operator. (a) In the presence of DNA, the anti-inducer forms a ternary complex with the repressor, primarily byestablishing hydrogen bonds between the O2 and O3 hydroxyls of the fucoside and of residues R197, N246, and D274 ofthe repressor, and the nitrophenyl group hydrogen bonds to N146. (b) In absence of DNA, the anti-inducer is also boundto the repressor by hydrogen bonding to the fucoside, but the nitrophenyl group does not appear to be ordered or to adoptthe same conformation.


two sequences resemble the primary operator,constitutive mutations were never found. On theother hand, these pseudo-operator sites increaserepression of the operon.58,59 The tetrameric repres-sor, in principal, is ideally suited to bind simulta-neously two of these operators and to createrepression loops that may play a role in regulation.60

Models for looping the DNA are consistent with thearchitecture of the tetrameric lac repressor that mayact similar to a clamp, bringing two operators thatare separated in linear sequence close together.61

Conclusion

Regulating gene expression is a fundamentalprocess of life and is essential for controllingmetabolic events, development, and disease. Al-though the specific details of regulation can beextraordinarily diverse and complex, the novelconcepts put forward by Jacob and Monod wererevolutionary and have served as the foundation forunderstanding gene regulation. Over the past halfcentury, the details of the operon have beenelucidated using genetic, biochemical, and structur-al techniques, yet the principles that were originallyput forward have only been slightly altered orrefined. The structures of these two repressors

provide detailed molecular models that help usunderstand how proteins recognize DNA and howthe synthesis of proteins is regulated.Many transcription regulators, in both prokaryotes

and eukaryotes, activate and repress the transcriptionof genes by recruitment.62 The structures of λrepressor beautifully illustrate how a protein boundto one site on the DNA can recruit a second protein tobind another site. When λ repressor binds to anoperator, a second repressor is recruited to an adjacentsite. The recruitment is driven by adhesive forces thatare created by protein–protein interactions. A repres-sor bound to the adjacent operator can then recruitpolymerase to bind its promoter. The same weakinteractions that are responsible of bringing repressormolecules together at adjacent sites are also respon-sible for recruiting repressors bound to operators thatare widely spaced along the genome. Recruitment oftranscriptional regulators is essential for maintainingthe functionality of this switch. Both positive andnegative regulation is achieved by simple adhesiveinteractions. Thermodynamically, recruitment andcooperativity are a consequence of linkedequilibrium.The diauxic growth of bacterial cultures described

by Monod in his doctoral thesis could not beexplained by the operon model.63 The operon isalso positively activated by a cyclic AMP-dependentcatabolite gene regulator protein [catabolite activator

Fig. 10. A summary of the genetic data for mutations that produce the Is phenotype. Each amino acid replacement wastested for β-galactosidase activity in the presence of IPTG. The genetic data are mapped on the surface structure of lacrepressor. Locations of mutants are shown in yellow and in red. Yellow areas are within 8 Å of the IPTG molecule.


protein (CAP).64 In glucose-starved cells, the level ofcyclic AMP increases dramatically, which in turnbinds to CAP. Once CAP is activated, the proteinbinds to the DNA, increasing the affinity of RNApolymerase for its promoter and transcription of thestructural genes of the lac operon.65 Transcription ofthe enzymes necessary for lactosemetabolism occurswhen the glucose levels in the cell are low and whenlactose is elevated. Although the detailed mecha-nism for alternate metabolite oxidation is likely to bemore complex, to a first approximation, this combi-nation of repression and activation can account forthe diauxie.The model of the operon has molded our

understanding of gene regulation and opened upnew and exciting areas of research. The regulatory

components of the lac operon have been used tocontrol transcription of the tyrosinase gene in amouse.66 In the absence of the inducer, the tyrosi-nase gene is repressed, and the mice have a whitecoat color. When the mice were fed IPTG in theirdrinking water, repression of tyrosinase was re-lieved, resulting in mice with brown eyes and fur.This simple bacterial switch successfully controlleda gene in a mammalian system. In the future, it iscertainly plausible that specific loci could beswitched on and off repeatedly to create reversiblemodels of human disease and normal development.As the mouse is the most widely used experimentalsystem to model human disease and development,the ability to regulate genes could greatly broadenthe range of biological questions that can be

image of Fig. 10


addressed experimentally. Monod would not havebeen at all surprised to see lac in mice, as he oncewrote, “anything that is true of E. colimust be true ofelephants, except more so.”

References

1. Jacob, F. & Monod, J. (1961). Genetic regulatorymechanisms in the synthesis of proteins. J. Mol. Biol.3, 318–356.

2. Monod, J. (1942). Recherches sur la croissance descultures bacteriennes. Hermann Editions Paris, Paris,France.

3. Judson, H. F. (1979). The Eighth Day of Creation: Makersof the Revolution in Biology. Simon and Schuster, NewYork, NY.

4. Cohn, M. (1957). Contributions of studies on the β-galactosidase of Escherichia coli to our understandingof enzyme synthesis. Bacteriol. Rev. 274, 765–769.

5. Lwoff, A. (1953). Lysogeny. Bacteriol. Rev. 17, 269–337.6. Lederberg, J. & Tatum, E. L. (1946). Gene recombina-

tion in Escherichia coli. Nature, 158, 558.7. Jacob, F. & Wollman, E. (1954). [Spontaneous induc-

tion of the development of bacteriophage λ duringgenetic recombination in Escherichia coli K12]. C. R.Hebd. Seances Acad. Sci. 239, 317–319.

8. Pardee, A. B., Jacob, F. & Monod, J. (1959). The geneticcontrol and cytoplasmic expression of “inducibility”in the synthesis of β-galactosidase by E. coli. J. Mol.Biol. 1, 165–178.

9. Gilbert,W. &Muller-Hill, B. (1966). Isolation of the LacRepressor. Proc. Natl Acad. Sci. USA, 56, 1891–1898.

10. Ptashne, M. (1967). Isolation of the λ phage repressor.Proc. Natl Acad. Sci. USA, 57, 306–313.

11. Ptashne, M. (1967). Specific binding of the λ phagerepressor to λ DNA. Nature, 214 , 232–234.

12. Gilbert, W. &Muller-Hill, B. (1967). The lac operator isDNA. Proc. Natl Acad. Sci. USA, 58, 2415–2421.

13. Muller-Hill, B., Crapo, L. & Gilbert, W. (1968).Mutants that make more lac repressor. Proc. NatlAcad. Sci. USA, 59, 1259–1264.

14. Beyreuther, K., Adler, K., Geisler, N. & Klemm, A.(1973). The amino-acid sequence of lac repressor. Proc.Natl Acad. Sci. USA, 70, 3576–3580.

15. Beyreuther, K. (1978). Revised sequence for the lacrepressor. Nature, 274, 767.

16. Geisler, N. & Weber, K. (1977). Isolation of amino-terminal fragment of lactose repressor necessary forDNA binding. Biochemistry, 16, 938–943.

17. Pabo, C. O., Sauer, R. T., Sturtevant, J. M. & Ptashne,M. (1979). The lambda repressor contains twodomains. Proc. Natl Acad. Sci. USA, 76, 1608–1612.

18. Jacob, F. &Monod, J. (1961). On the regulation of geneactivity. Cold Spring Harbor Symp. Quant. Biol. 26,193–211.

19. Bourgeois, S. & Riggs, A. D. (1970). The lac repressor–operator interaction. IV. Assay and purification ofoperator DNA. Biochem. Biophys. Res. Commun. 38,348–354.

20. Gilbert, W. & Maxam, A. (1973). The nucleotidesequence of the lac operator. Proc. Natl Acad. Sci.USA, 70, 3581–3584.

21. Bahl, C. P., Wu, R., Stawinsky, J. & Narang, S. A.(1977). Studies on the lactose operon. Minimal lengthof the lactose operator sequence for the specificrecognition by the lactose repressor. Proc. Natl Acad.Sci. USA, 74, 966–970.

22. Ptashne, M. & Hopkins, N. (1968). The operatorscontrolled by the λ phage repressor. Proc. Natl Acad.Sci. USA, 60, 1282–1287.

23. Maniatis, T. & Ptashne, M. (1973). Multiple repressorbinding at the operators in bacteriophage λ. Proc. NatlAcad. Sci. USA, 70, 1531–1535.

24. Seeman, N. C., Rosenberg, J. M. & Rich, A. (1976).Sequence-specific recognition of double helical nucleicacids by proteins. Proc. Natl Acad. Sci. USA, 73, 804–808.

25. Pabo, C. O. & Lewis, M. (1982). The operator-bindingdomain of λ repressor: structure and DNA recogni-tion. Nature, 298, 443–447.

26. Beamer, L. J. & Pabo, C. O. (1992). Refined 1.8 Åcrystal structure of the λ repressor–operator complex.J. Mol. Biol. 227, 177–196.

27. Jordan, S. R. & Pabo, C. O. (1988). Structure of thelambda complex at 2.5 Å resolution: details of therepressor–operator interactions. Science, 242, 893–899.

28. Chuprina, V. P., Rullmann, J. A., Lamerichs, R.M., vanBoom, J. H., Boelens, R. & Kaptein, R. (1993). Structureof the complex of lac repressor headpiece and an 11base-pair half-operator determined by nuclear mag-netic resonance spectroscopy and restrainedmoleculardynamics. J. Mol. Biol. 234 , 446–462.

29. Sauer, R. T., Yocum, R. R., Doolittle, R. F., Lewis, M. &Pabo, C. O. (1982). Homology among DNA-bindingproteins suggests use of a conserved super-secondarystructure. Nature, 298, 447–451.

30. Friedman, A. M., Fischmann, T. O. & Steitz, T. A.(1995). Crystal structure of lac repressor core tetramerand its implications for DNA looping. Science, 268,1721–1727.

31. Bell, C. E. & Lewis, M. (2001). Crystal structure of theλ repressor C-terminal domain octamer. J. Mol. Biol.314, 1127–1136.

32. Bell, C. E., Frescura, P., Hochschild, A. & Lewis, M.(2000). Crystal structure of the λ repressor C-terminaldomain provides a model for cooperative operatorbinding. Cell, 101, 801–811.

33. Lewis, M., Chang, G., Horton, N. C., Kercher, M. A.,Pace, H. C., Schumacher, M. A. et al. (1996). Crystalstructure of the lactose operon repressor and itscomplexes with DNA and inducer. Science, 271,1247–1254.

34. Stayrook, S., Jaru-Ampornpan, P., Ni, J., Hochschild,A. & Lewis, M. (2008). Crystal structure of the λrepressor and a model for pairwise cooperativeoperator binding. Nature, 452, 1022–1025.

35. Johnson, A. D., Meyer, B. J. & Ptashne, M. (1979).Interactions between DNA-bound repressors governregulation by the λ phage repressor. Proc. Natl Acad.Sci. USA, 76, 5061–5065.

36. Benson, N., Adams, C. & Youderian, P. (1994). Geneticselection for mutations that impair the co-operativebinding of lambda repressor. Mol. Microbiol. 11,567–579.

37. Ackers, G. K., Johnson, A. D. & Shea, M. A. (1982).Quantitative model for gene regulation by λ phagerepressor. Proc. Natl Acad. Sci. USA, 79, 1129–1133.


38. Beckett, D., Burz, D. S., Ackers, G. K. & Sauer, R. T.(1993). Isolation of λ repressor mutants with defects incooperative operator binding. Biochemistry, 32,9073–9079.

39. Whipple, F. W., Hou, E. F. & Hochschild, A. (1998).Amino acid–amino acid contacts at the cooperativityinterface of the bacteriophage λ and P22 repressors.Genes Dev. 12, 2791–2802.

40. Ptashne, M., Backman, K., Humayun, M. Z., Jeffrey,A., Maurer, R., Meyer, B. & Sauer, R. T. (1976).Autoregulation and function of a repressor in bacte-riophage lambda. Science, 194, 156–161.

41. Hochschild, A. & Ptashne, M. (1986). Cooperativebinding of λ repressors to sites separated by integralturns of the DNA helix. Cell, 44, 681–687.

42. Dodd, I. B. & Egan, J. B. (2002). Action at a distance inCI repressor regulation of the bacteriophage 186genetic switch. Mol. Microbiol. 45, 697–710.

43. Slilaty, S. N. & Little, J. W. (1987). Lysine-156 andserine-119 are required for LexA repressor cleavage: apossible mechanism. Proc. Natl Acad. Sci. USA, 84,3987–3991.

44. Sauer, R. T., Ross, M. J. & Ptashne, M. (1982). Cleavageof the λ and P22 repressors by recA protein. J. Biol.Chem. 257, 4458–4462.

45. Dodson, G. & Wlodawer, A. (1998). Catalytic triadsand their relatives. Trends Biochem. Sci. 23, 347–352.

46. Little, J. W. & Mount, D. W. (1982). The SOSregulatory system of Escherichia coli. Cell, 29, 11–22.

47. Gimble, F. S. & Sauer, R. T. (1985). Mutations inbacteriophage λ repressor that prevent RecA-mediatedcleavage. J. Bacteriol. 162, 147–154.

48. Monod, J., Changeux, J. P. & Jacob, F. (1963). Allostericproteins and cellular control systems. J. Mol. Biol. 6,306–329.

49. Monod, J., Wyman, J. & Changeux, J. P. (1965). On thenature of allosteric transitions: a plausiblemodel. J. Mol.Biol. 12, 88–118.

50. Pace, H. C., Lu, P. & Lewis, M. (1990). lac repressor:crystallization of intact tetramer and its complexeswith inducer and operator DNA. Proc. Natl Acad. Sci.USA, 87, 1870–1873.

51. Haurowitz, F. (1938). Hoppe-Seyler's Z. Physiol. Chem.254, 268–274.

52. Daber, R., Sharp, K. & Lewis, M. (2009). One is notenough. J. Mol. Biol. 392, 1133–1144.

53. Daber, R., Stayrook, S., Rosenberg, A. & Lewis, M.(2007). Structural analysis of lac repressor bound toallosteric effectors. J. Mol. Biol. 370, 609–619.

54. Markiewicz, P., Kleina, L. G., Cruz, C., Ehret, S. &Miller, J. H. (1994). Genetic studies of the lac repressor.XIV. Analysis of 4000 altered Escherichia coli lacrepressors reveals essential and non-essential resi-dues, as well as “spacers” which do not require aspecific sequence. J. Mol. Biol. 240, 421–433.

55. Schlax, P. J., Capp, M. W. & Record, M. T., Jr (1995).Inhibition of transcription initiation by lac repressor.J. Mol. Biol. 245, 331–350.

56. Riggs, A. D., Bourgeois, S. & Cohn, M. (1970). The lacrepressor–operator interaction. 3. Kinetic studies. J. Mol.Biol. 53, 401–417.

57. Riggs, A. D., Bourgeois, S. & Cohn, M. (1970). The lacrepressor–operator interaction. J. Mol. Biol. 53, 401–417.

58. Mossing, M. & Record, M. T., Jr (1986). Upstreamoperators enhance repression of the lac promoter.Science, 233, 889–892.

59. Oehler, S., Eismann, E. R., Kramer, H. & Muller-Hill,B. (1990). The three operators of the lac operoncooperate in repression. EMBO J. 9, 973–979.

60. Kania, J. & Muller-Hill, B. (1977). Construction,isolation and implications of repressor–galactosidase—β-galactosidase hybrid molecules. Eur. J. Biochem.79, 381–386.

61. Swigon, D., Coleman, B. D. & Olson, W. K. (2006).Modeling the Lac repressor–operator assembly: theinfluence of DNA looping on Lac repressor confor-mation. Proc. Natl Acad. Sci. USA, 103, 9879–9884.

62. Ptashne, M. (2005). Regulation of transcription: fromlambda to eukaryotes. Trends Biochem. Sci. 30, 275–279.

63. Makman, R. S. & Sutherland, E. W. (1965). Adenosine3′,5′-phosphate in Escherichia coli. J. Biol. Chem. 240,1309–1314.

64. Ullmann, A. & Monod, J. (1968). Cyclic AMP as anantagonist of catabolite repression in Escherichia coli.FEBS Lett. 2 , 57–60.

65. Zubay, G., Schwartz, D. & Beckwith, J. (1970).Mechanism of activation of catabloite-sensitivegenes: a positive control. Proc. Natl Acad. Sci. USA,66, 104–110.

66. Cronin, C. A., Gluba, W. & Scrable, H. (2001). The lacoperator–repressor system is functional in the mouse.Genes Dev. 15, 1506–1517.

A Tale of Two Repressors

Documents

Transcript of A Tale of Two Repressors