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You are watching: The product of the laci gene is _____.
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.
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A combination of genetic and biochemical experiments in bacteria led to the initialrecognition of (1) protein-binding regulatory sequences associated with genes and(2) proteins whose binding to a gene’s regulatory sequences eitheractivate or repress its transcription. These key components underlie the ability ofboth prokaryotic and eukaryotic cells to turn genes on and off, although innumerablevariations on the basic process have been discovered. In this section, we firstdescribe some of the early experimental findings leading to a general model ofbacterial transcription control. In the next section, we take a closer look at howbacterial RNA polymerase initiates transcription and the mechanisms controlling itsability to do so.
In bacteria, gene control serves mainly to allow a single cell to adjust to changesin its nutritional environment so that its growth and division can be optimized.Thus, the prime focus of research has been on genes that encodeinducible proteins whose production varies depending on thenutritional status of the cells. Although gene control in multicellular organismsoften involves response to environmental changes, its most characteristic andbiologically far-reaching purpose is the regulation of a genetic program thatunderlies embryological development and tissue differentiation. Nonetheless, many ofthe principles of transcription control first discovered in bacteria also apply toeukaryotic cells.
Enzymes Encoded at the lac Operon Can Be Induced andRepressed
E. coli can use either glucose or other sugars such as thedisaccharide lactose as the sole source of carbon and energy. When E.coli cells are grown in a glucose-containing medium, the activityof the enzymes needed to metabolize lactose is very low. When these cells areswitched to a medium containing lactose but no glucose, the activities of thelactose-metabolizing enzymes increase. Early studies showed that the increase inthe activity of these enzymes resulted from the synthesis of new enzymemolecules, a phenomenon termed induction. The enzymes induced in the presence of lactose areencoded by the lac operon, which includes two genes,Z and Y, that are required for metabolismof lactose and a third gene, A (Figure 10-1). The lacY gene encodeslactose permease, which spans the E. colicell membrane and uses the energy available from the electrochemical gradientacross the membrane to pump lactose into the cell (Section 15.5). ThelacZ gene encodesβ-galactosidase, which splits the disaccharidelactose into the monosaccharides glucose and galactose (see Figure 2-10); these sugars are further metabolized throughthe action of enzymes encoded in other operons. The lacA geneencodes thiogalactoside transacetylase, an enzyme whosephysiological function is not well understood.
The lac operon includes three genes:lacZ, which encodes β-galactosidase;lacY, which encodes lactose permease; andlacA, which encodes thiogalactosidetransacetylase. Binding of regulatory proteins to sites in the control regionimmediately upstream (more…)
Synthesis of all three enzymes encoded in the lac operon israpidly induced when E. coli cells are placed in a mediumcontaining lactose as the only carbon source and repressed when the cells areswitched to a medium without lactose. Thus all three genes of thelac operon are coordinately regulated. Thelac operon in E. coli provides one of theearliest and still best-understood examples of gene control. Much of thepioneering research on the lac operon was conducted by FrancoisJacob, Jacques Monod, and their colleagues in the 1960s.
Some molecules similar in structure to lactose can induce expression of thelac-operon genes even though they cannot be hydrolyzed byβ-galactosidase. Such small molecules (i.e., smaller than proteins) arecalled inducers. One of these,isopropyl-β-D-thiogalactoside, abbreviated IPTG,isparticularly useful in genetic studies of the lac operon,because it can diffuse into cells and, since it is not metabolized, itsconcentration remains constant throughout an experiment.
Mutations in lacI Cause Constitutive Expression oflac Operon
Insight into the mechanisms controlling synthesis of β-galactosidase andlactose permease first came from the study of mutants in which control ofβ-galactosidase expression was abnormal. A sensitive colorimetric assayfor β-galactosidase uses X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) as substrate:
Hydrolysis of this colorless analogof lactose by β-galactosidase yields an intensely blue product. Whenwild-type E. coli cells are plated on media containing X-galplus lactose as the major carbon source, all the colonies that grow appear blue.When the cells are plated on media containing X-gal plus glucose as the carbonsource, the resulting colonies appear white; in this case,β-galactosidase synthesis is repressed and there is not sufficientβ-galactosidase in the cells to hydrolyze the X-gal to its coloredproduct. However, when the cells are exposed to chemical mutagens before platingon X-gal/glucose plates, rare blue colonies appear. In most cases, when cellsfrom these blue colonies are recovered and grown in media containing glucose,they are found to express all the genes of the lac operon atmuch higher levels than wild-type cells in the same medium. Such cells arecalled constitutive mutants becausethey fail to repress the lac operon in media lacking lactoseand instead continuously, or constitutively, express the enzymes. Byrecombinational analysis (Section 8.3), these mutations were mapped to a regionon the E. coli chromosome to the left of thelacZ gene, a region called the lacI gene(see Figure 10-1).
Jacob and Monod reasoned that such constitutive mutants probably had a defect ina protein that normally repressed expression of the lac operonin the absence of lactose. Hence they called the protein encoded by thelacI gene the lac repressor and proposedthat it binds to a site on the E. coli genome wheretranscription of the lac operon is initiated, thereby blockingtranscription. They further hypothesized that when lactose is present in thecell, it binds to the lac repressor, decreasing its affinityfor the repressor-binding site on the DNA. As a result, the repressor falls offthe DNA and transcription of the lac operon is initiated,leading to synthesis of β-galactosidase, lactose permease, andthiogalactoside transacetylase (Figure10-2).
Jacob and Monod model of transcriptional regulation of thelac operon by lacrepressor. When lac repressor binds to a DNA sequence calledthe operator (O), which lies just upstream of thelacZ gene, transcription of the operon by RNA polymerase is blocked. (more…)
Isolation of Operator Constitutive and Promoter Mutants Support Jacob-MonodModel
The model proposed by Jacob and Monod predicted that a specific DNA sequence nearthe transcription start site of the lac operon is a bindingsite for lac repressor. They reasoned that mutations in thissequence, which they termed the operator (O), would prevent the repressor from binding, thusyielding constitutive mutants that could be identified on X-gal/glucoseindicator plates. To distinguish between mutations in the lacIgene, which inactivate the repressor, and mutations in the operator, whichprevent repressor binding, Jacob and Monod mutagenized cells carrying two copiesof the wild-type lacI gene, one on the bacterial chromosome andone on a plasmid. In this system, separate mutations in both copies oflacI in a given cell are required to generate alacI− constitutive mutant, alow-probability event. In contrast, only a single mutation in the operator ofone copy of the lac operon is required to yield a constitutivemutant. Using this approach, Jacob and Monod isolated mutants that expressed thelac operon constitutively even when two copies of thewild-type lacI gene encoding the lac repressorwere present in the same cell. These operator constitutive(Oc) mutations mapped to one end of thelac operon, as the model predicted (see Figure 10-2).
Most mutations that prevent expression of β-galactosidase in cellsexposed to an inducer such as IPTG map in the lacZ gene itself.But a rare class of mutations map to a region between lacI andthe operator, in a region termed the promoter (P). Cells carrying these mutations also cannot induceexpression of the lacY and lacA genes; thatis, these mutations prevent expression of the entire lacoperon. According to the Jacob and Monod model, such promoter mutations blockinitiation of transcription by RNA polymerase (see Figure 10-2). Consequently, no lac mRNAand therefore no lac proteins are synthesized, even whenlac repressor binds IPTG and comes off thelac operator.
Regulation of lac Operon Depends on Cis-Acting DNA Sequencesand Trans-Acting Proteins
Subsequent analyses of the effects of various mutations in E.coli cells containing one or two copies of lac DNAprovided further insight into regulation of lac-operonexpression. In these experiments, assays for β-galactosidase andlactose permease activity were conducted in the presence and absence of inducer(IPTG). These analyses showed that the Oc mutation is dominant overO+ (the wild-type lac O sequence). Inaddition, the Oc mutation only affects expression oflac genes on the same DNA molecule (i.e., genes in cis tothe mutation). Experimental demonstration of the cis-acting nature of the Oc mutation isillustrated in Figure 10-3.
Experimental demonstration that Oc mutations arecis-acting. E. coli cells containing two copies of thelac operon are diagrammed. Diagonal linesindicate genes and control regions carrying mutations. In thesecells, the lac operon on the bacterial chromosomehas (more…)
As noted earlier, mutations in lacI (in cells with a singlelac operon) cause constitutive expression ofβ-galactosidase and lactose permease because no functional repressor ismade. Unlike the Oc mutation, which is dominant, thelacI− mutation is recessive to thewild-type lacI+ gene. Furthermore, thewild-type lacI+ gene can exert control overthe lacZ and lacY genes on a different DNAmolecule (i.e., genes in trans to lacI+).The trans-acting ability oflacI+is easy to understand since thisgene encodes a protein, which is free to diffuse through the cell and bind toany lac operator in the cell (Figure 10-4).
Experimental demonstration that thelacI+ gene istrans-acting. (Top) Cells carrying a singlelacI− gene produce aninactive repressor; as a result, they expressβ-galactosidase and lactose permease constitutively.(Bottom) When a wild-type (more…)
In general, cis-acting mutations are in DNA sequences that function as bindingsites for proteins that control the expression of nearby genes. For example, thecis-acting Oc mutations prevent binding of the lacrepressor to the operator. Similarly, mutations in the lacpromoter are cis-acting, since they alter the binding site for RNA polymerase.When RNA polymerase cannot initiate transcription of the lacoperon, none of the genes in the operon can be expressed irrespective of thefunction of the repressor. In general, trans-acting genes that regulateexpression of genes on other DNA molecules encode diffusible products. In mostcases these are proteins, but in some cases RNA molecules can act in trans toregulate gene expression.
Biochemical Experiments Confirm That Induction of the lacOperon Leads to Increased Synthesis of lac mRNA
The Jacob and Monod model of repressor control of lac operontranscription, which was based on genetic experiments with E.coli mutants, proposes that addition of inducer causes an increasein transcription of the lac operon. This prediction was testeddirectly through pulse-labeling experiments that measured the rate oflac mRNA synthesis in E. coli cells growninitially in glucose media and then after addition of IPTG. The results of suchexperiments showed that little lac mRNA is synthesized beforethe addition of IPTG, but lac mRNA synthesis is detectablewithin 1 minute after the addition of IPTG and reaches a maximal rate by 2minutes (Figure 10-5). At later times,lac mRNA synthesis is maintained at this maximal rate aslong as inducer is present. These findings demonstrated directly that inducerdoes indeed cause an increase in transcription of the lacoperon.
Biochemical demonstration that inducer leads to an increase in lac operon transcription. Small samples of an E. coli culture growing inglucose medium were removed just before and at short intervals afteraddition of IPTG. <3H>uridine was added to each (more…)
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