Chapter 26The Operon
26.1 Introduction
•coupled transcription/translation – The phenomena inbacteria where translation of the mRNA occurssimultaneously with its transcription.
•operon – A unit of bacterial gene expression andregulation, including structural genes and controlelements in DNA recognized by regulator geneproduct(s).
26.1 Introduction
•trans-acting – A product that can function on any copyof its target DNA. This implies that it is a diffusible proteinor RNA.
•cis-acting – A site that affects the activity only ofsequences on its own molecule of DNA (or RNA); thisproperty usually implies that the site does not code forprotein.
26.1 Introduction
•regulator gene – A gene that codes for a product(typically protein) that controls the expression of othergenes (usually at the level of transcription).
•structural gene – A gene that codes for any RNA orprotein product other than a regulator.
FIGURE 01: A regulatorbinds a target site on DNA
26.1 Introduction
•In negative regulation, a repressor protein binds to anoperator to prevent a gene from being expressed.
•In positive regulation, a transcription factor is requiredto bind at the promoter in order to enable RNApolymerase to initiate transcription.
FIGURE 02: A repressor stopsRNA polymerase from initiating
FIGURE 03: Transcription factors enableRNA polymerase to bind to the promoter
26.1 Introduction
•In inducible regulation, the gene is regulated by thepresence of its substrate (the inducer).
•In repressible regulation, the gene is regulated by theproduct of its enzyme pathway (the corepressor).
26.1 Introduction
•We can combine these inall four combinations:negative inducible,negative repressible,positive inducible, andpositive repressible.
FIGURE 04: Induction andrepression can be under positiveor negative control
26.2 Structural Gene Clusters AreCoordinately Controlled
•Genes coding for proteins that function in the samepathway may be located adjacent to one another andcontrolled as a single unit that is transcribed into apolycistronic mRNA.
FIGURE 05: The lac operon includes cis-acting regulator elements andprotein-coding structural genes
26.3 The lac Operon Is Negative Inducible
•Transcription of the lacZYAoperon is controlled by arepressor protein (the lacrepressor) that binds to anoperator that overlaps thepromoter at the start of thecluster.
•constitutive expression –A state in which a gene isexpressed continuously.
•In the absence of β-galactosides, the lac operonis expressed only at a verylow (basal) level.
FIGURE 06: The promoter andoperator overlap
26.3 The lac Operon Is Negative Inducible
•The repressor protein is a tetramer of identical subunitscoded by the lacI gene.
•β-galactoside sugars, the substrates of the lac operon,are its inducer.
•Addition of specific β-galactosides induces transcriptionof all three genes of the lac operon.
•The lac mRNA is extremely unstable; as a result,induction can be rapidly reversed.
FIGURE 07: lac expression responds to inducer
26.4 lac Repressor Is Controlled by aSmall-Molecule Inducer
•An inducer functions byconverting the repressorprotein into a form with loweroperator affinity.
•Repressor has two bindingsites, one for the operator DNAand another for the inducer.
•gratuitous inducer – Inducersthat resemble authenticinducers of transcription, butare not substrates for theinduced enzymes.
FIGURE 08: A repressortetramer binds the operator toprevent transcription
26.4 lac Repressor Is Controlled by aSmall-Molecule Inducer
•Repressor is inactivated by an allosteric interaction in which bindingof inducer at its site changes the properties of the DNA-binding site(allosteric control).
•The true inducer is allolactose, not the actual substrate of β-galactosidase.
FIGURE 09: Inducer inactivatesrepressor, allowing gene expression
26.5 cis-Acting Constitutive MutationsIdentify the Operator
•Mutations in the operator cause constitutive expressionof all three lac structural genes.
•These mutations are cis-acting and affect only thosegenes on the contiguous stretch of DNA.
•Mutations in the promoter prevent expression of lacZYAare uninducible and cis-acting.
26.5 cis-Acting Constitutive MutationsIdentify the Operator
•cis-dominant – A site or mutation that affects the properties only ofits own molecule of DNA, often indicating that a site does not codefor a diffusible product.
FIGURE 10: Constitutive operatormutant cannot bind repressor protein
26.6 trans-Acting Mutations Identify theRegulator Gene
•Mutations in the lacI gene are trans-acting and affectexpression of all lacZYA clusters in the bacterium.
•Mutations that eliminate lacI function cause constitutiveexpression and are recessive (lacI–).
•Mutations in the DNA-binding
site of the repressor are
constitutive because the
repressor cannot bind the
operator.
FIGURE 11: Defective repressorcauses constitutive expression
26.6 trans-Acting Mutations Identify theRegulator Gene
•Mutations in the inducer-binding site of therepressor prevent it from being inactivated andcause uninducibility.
•When mutant and wild-type subunits arepresent, a single lacI–d mutant subunit caninactivate a tetramer whose other subunits arewild-type.
–It is dominant negative.
26.6 trans-Acting Mutations Identify theRegulator Gene
•interallelic complementation – Thechange in the properties of aheteromultimeric protein brought about bythe interaction of subunits coded by twodifferent mutant alleles.
–The mixed protein may be more or less active thanthe protein consisting of subunits of only one or theother type.
26.6 trans-Acting Mutations Identify theRegulator Gene
•negative complementation– This occurs when interalleliccomplementation allows amutant subunit to suppressthe activity of a wild-typesubunit in a multimericprotein.
•lacI–d mutations occur in theDNA-binding site. Their effectis explained by the fact thatrepressor activity requires allDNA-binding sites in thetetramer to be active.
FIGURE 12: Negativecomplementation identifies proteinmultimer
26.7 lac Repressor Is a Tetramer Made ofTwo Dimers
•A single repressor subunit can be divided into the N-terminal DNA-binding domain, a hinge, and the core ofthe protein.
•The DNA-binding domain contains two short α-helicalregions that bind the major groove of DNA.
•The inducer-binding site and the regions responsible formultimerization are located in the core.
FIGURE 13: Lac repressor monomer has several domains
Structure from Protein Data Bank 1LBG. M. Lewis, et al., Science 271(1996): 1247-1254. Photo courtesy of Hongli Zhan and Kathleen S.Matthews, Rice University.
26.7 lac Repressor Is a Tetramer Made ofTwo Dimers
•Monomers form a dimer bymaking contacts betweencore subdomains 1 and 2.
•Dimers form a tetramer byinteractions between thetetramerization helices.
FIGURE 15: Repressor is a tetramerof two dimers
26.7 lac Repressor Is a Tetramer Made ofTwo Dimers
•Different types of mutationsoccur in different domainsof the repressor protein.
FIGURE 16: Mutations identifyrepressor domains
26.8 lac Repressor Binding to the OperatorIs Regulated by an Allosteric Change inConformation
•lac repressor protein binds to the double-stranded DNAsequence of the operator.
•The operator is a palindromic sequence of 26 bp.
•Each inverted repeat of the operator binds to the DNA-binding site of one repressor subunit.
FIGURE 17: The lac operator has dyad symmetry
26.8 lac Repressor Binding to the OperatorIs Regulated by an Allosteric Change inConformation
•Inducer binding causes achange in repressorconformation that reduces itsaffinity for DNA and releasesit from the operator.
FIGURE 18: Inducer controlsrepressor conformation
26.9 lac Repressor Binds to ThreeOperators and Interacts with RNAPolymerase
•Each dimer in a repressor tetramer can bind an operator,so that the tetramer can bind two operatorssimultaneously.
•Full repression requires the repressor to bind to anadditional operator downstream or upstream as well asto the primary operator at the lacZ promoter.
•Binding of repressor at the operator stimulates binding ofRNA polymerase at the promoter but precludestranscription.
FIGURE 21: Repressor can make aloop in DNA
26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor
•Proteins that have a high affinity for a specific DNAsequence also have a low affinity for other DNAsequences.
•Every base pair in the bacterial genome is the start of alow-affinity binding site for repressor.
FIGURE 23: Repressor specifically binds operator DNA
26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor
•The large number of low-affinity sites ensures that allrepressor protein is boundto DNA.
•Repressor binds to theoperator by moving from alow-affinity site rather thanby equilibrating fromsolution.
FIGURE 24: Repression affectsthe sites at which repressor isbound on DNA
26.10 The Operator Competes with Low-Affinity Sites to Bind Repressor
•In the absence of inducer, the operator has an affinity forrepressor that is 107 times that of a low-affinity site.
•The level of 10 repressor tetramers per cell ensures thatthe operator is bound by repressor 96% of the time.
•Induction reduces the affinity for the operator to 104times that of low-affinity sites, so that operator is boundonly 3% of the time.
26.11 The lac Operon Has a Second Layerof Control: Catabolite Repression
•catabolite repression – The ability of glucoseto prevent the expression of a number of genes.
–In bacteria this is a positive control system; ineukaryotes, it is completely different.
•Catabolite repressor protein (CRP) is anactivator protein that binds to a target sequenceat a promoter.
FIGURE 25: CRP binds to a consensus sequence.
26.11 The lac Operon Has a Second Layerof Control: Catabolite Repression
•A dimer of CRP is activated bya single molecule of cyclicAMP (cAMP).
•cAMP is controlled by thelevel of glucose in the cell; alow glucose level allowscAMP to be made.
•CRP interacts with the C-terminal domain of the αsubunit of RNA polymerase toactivate it.
FIGURE 27: Glucose reducesCRP activity
26.12 The trp Operon Is a RepressibleOperon with Three Transcription Units
•The trp operon is negatively controlled by the level of itsproduct, the amino acid tryptophan (autoregulation).
•The amino acid tryptophan activates an inactiverepressor encoded by trpR.
•A repressor (or activator) will act on all loci that have acopy of its target operator sequence.
FIGURE 30: CRP-binding sites areclose to the promoter
26.13 The trp Operon Is Also Controlled byAttenuation
•attenuation – The regulation of bacterial operons bycontrolling termination of transcription at a site locatedbefore the first structural gene.
FIGURE 33: Termination canbe controlled via changes inRNA secondary structure
26.13 The trp Operon Is Also Controlled byAttenuation
•An attenuator (intrinsic terminator) is located betweenthe promoter and the first gene of the trp cluster.
•The absence of Trp-tRNA suppresses termination andresults in a 10 increase in transcription.
FIGURE 34: An attenuatorcontrols progression of RNApolymerase into trp genes
26.14 Attenuation Can Be Controlled byTranslation
•The leader region of the trp operon has a fourteen-codonopen reading frame that includes two codons fortryptophan.
•The structure of RNA at the attenuator depends onwhether this reading frame is translated.
•In the presence of Trp-tRNA, the leader is translated to aleader peptide, and the attenuator is able to form thehairpin that causes termination.
26.14 AttenuationCan Be Controlledby Translation
FIGURE 35: The trp operon has ashort sequence coding for aleader peptide
26.14 Attenuation Can Be Controlled byTranslation
FIGURE 36: The trp leader region canexist in alternative base-pairedconformations
FIGURE 37: Tryptophan controlsribosome position
26.14 Attenuation Can Be Controlled byTranslation
•In the absence of Trp-tRNA, the ribosome stalls at thetryptophan codons and an alternative secondarystructure prevents formation of the hairpin, so thattranscription continues.
FIGURE 38: Trp-tRNA controlsthe E. coli trp operon directly
26.15 Translation Can Be Regulated
•Translation can be regulated by the 5′ UTR of the mRNA.
•Translation may be regulated by the abundance of varioustRNAs (codon usage).
•A repressor protein can regulate translation by preventinga ribosome from binding to an initiation codon.
FIGURE 39: A regulator may blockribosome binding
26.15 Translation Can Be Regulated
•Accessibility of initiationcodons in a polycistronicmRNA can be controlled bychanges in the structure ofthe mRNA that occur as theresult of translation.
FIGURE 41: Ribosome movementcan control translation
26.16 r-Protein Synthesis Is Controlled byAutoregulation
•Translation of an r-protein operon can be controlled by aproduct of the operon that binds to a site on thepolycistronic mRNA.
FIGURE 43: rRNA controls thelevel of free r-proteins