H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 48
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In multicellular organisms, control of geneexpression is largely directed toward assuring that the rightgene is expressed in the right cell at the right time during embryological development and tissue differentiation. Here wedescribe the basic features of transcription control in bacteria, using the lac operon in E. coli as our primary example.Many of the same processes, as well as others, are involvedin eukaryotic transcription control, which is discussed inChapter 11.In E. coli, about half the genes are clustered into operons each of which encodes enzymes involved in a particularmetabolic pathway or proteins that interact to form one multisubunit protein.
For instance, the trp operon mentionedearlier encodes five enzymes needed in the biosynthesis oftryptophan (see Figure 4-12). Similarly, the lac operon encodes three enzymes required for the metabolism of lactose,a sugar present in milk. Since a bacterial operon is tran-115scribed from one start site into a single mRNA, all the geneswithin an operon are coordinately regulated; that is, they areall activated or repressed to the same extent.Transcription of operons, as well as of isolated genes, iscontrolled by an interplay between RNA polymerase andspecific repressor and activator proteins.
In order to initiatetranscription, however, E. coli RNA polymerase must be associated with one of a small number of (sigma) factors,which function as initiation factors. The most common onein bacterial cells is 70.Initiation of lac Operon TranscriptionCan Be Repressed and ActivatedWhen E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed, so that cellular energy isnot wasted synthesizing enzymes the cells cannot use.
In anenvironment containing both lactose and glucose, E. colicells preferentially metabolize glucose, the central moleculeof carbohydrate metabolism. Lactose is metabolized at ahigh rate only when lactose is present and glucose is largelydepleted from the medium. This metabolic adjustment isachieved by repressing transcription of the lac operon untillactose is present, and synthesis of only low levels of lacmRNA until the cytosolic concentration of glucose falls tolow levels. Transcription of the lac operon under differentconditions is controlled by lac repressor and catabolite activator protein (CAP), each of which binds to a specificDNA sequence in the lac transcription-control region (Figure 4-16, top).For transcription of the lac operon to begin, the 70 subunit of the RNA polymerase must bind to the lac promoter,which lies just upstream of the start site.
When no lactose ispresent, binding of the lac repressor to a sequence called thelac operator, which overlaps the transcription start site,blocks transcription initiation by the polymerase (Figure4-16a). When lactose is present, it binds to specific bindingsites in each subunit of the tetrameric lac repressor, causing aconformational change in the protein that makes it dissociatefrom the lac operator. As a result, the polymerase can initiatetranscription of the lac operon.
However, when glucose alsois present, the rate of transcription initiation (i.e., the numberof times per minute different polymerase molecules initiatetranscription) is very low, resulting in synthesis of only lowlevels of lac mRNA and the proteins encoded in the lacoperon (Figure 4-16b).Once glucose is depleted from the media and the intracellular glucose concentration falls, E. coli cells respond bysynthesizing cyclic AMP, cAMP (see Figure 3-27b). As theconcentration of cAMP increases, it binds to a site in eachsubunit of the dimeric CAP protein, causing a conformational change that allows the protein to bind to the CAP sitein the lac transcription-control region.
The bound CAPcAMP complex interacts with the polymerase bound to thepromoter, greatly stimulating the rate of transcription initiation. This activation leads to synthesis of high levels of lac116CHAPTER 4 • Basic Molecular Genetic Mechanisms+1 (transcription start site)PromoterlacZOperatorCAP siteE. coli lac transcription-control genesCAPPol-σ70(a)lac repressor− lactoselacZ+ glucose(low cAMP)No mRNA transcriptionlactose(b)+ lactoselacZ+ glucose(low cAMP)(c)+ lactose− glucose(high cAMP)rate of initiation is further reduced by the lac repressor andsubstantially increased by the cAMP-CAP activator.Low transcriptioncAMPlacZHigh transcription▲ FIGURE 4-16 Regulation of transcription from thelac operon of E. coli.
(Top) The transcription-control region,composed of ≈100 base pairs, includes three protein-bindingregions: the CAP site, which binds catabolite activator protein;the lac promoter, which binds the RNA polymerase–70 complex;and the lac operator, which binds lac repressor. The lacZ gene,the first of three genes in the operon, is shown to the right.(a) In the absence of lactose, very little lac mRNA is producedbecause the lac repressor binds to the operator, inhibitingtranscription initiation by RNA polymerase–70. (b) In thepresence of glucose and lactose, lac repressor binds lactoseand dissociates from the operator, allowing RNA polymerase–70to initiate transcription at a low rate. (c) Maximal transcription ofthe lac operon occurs in the presence of lactose and absence ofglucose. In this situation, cAMP increases in response to the lowglucose concentration and forms the CAP-cAMP complex, whichbinds to the CAP site, where it interacts with RNA polymeraseto stimulate the rate of transcription initiation.mRNA and subsequently of the enzymes encoded by the lacoperon (Figure 4-16c).Although the promoters for different E.
coli genes exhibitconsiderable homology, their exact sequences differ. The promoter sequence determines the intrinsic rate at which anRNA polymerase– complex initiates transcription of a genein the absence of a repressor or activator protein. Promotersthat support a high rate of transcription initiation are calledstrong promoters. Those that support a low rate of transcription initiation are called weak promoters. The lacoperon, for instance, has a weak promoter; its low intrinsicSmall Molecules Regulate Expression of ManyBacterial Genes via DNA-Binding RepressorsTranscription of most E.
coli genes is regulated by processessimilar to those described for the lac operon. The generalmechanism involves a specific repressor that binds to the operator region of a gene or operon, thereby blocking transcription initiation. A small molecule (or molecules), called aninducer, binds to the repressor, controlling its DNA-bindingactivity and consequently the rate of transcription as appropriate for the needs of the cell.For example, when the tryptophan concentration in themedium and cytosol is high, the cell does not synthesize theseveral enzymes encoded in the trp operon. Binding of tryptophan to the trp repressor causes a conformational changethat allows the protein to bind to the trp operator, therebyrepressing expression of the enzymes that synthesize tryptophan. Conversely, when the tryptophan concentration in themedium and cytosol is low, tryptophan dissociates from thetrp repressor, causing a conformational change in the proteinthat causes it to dissociate from the trp operator, allowingtranscription of the trp operon.
In the case of the lac operon,binding of the inducer lactose to the lac repressor reducesbinding of the repressor to the operator, thereby promotingtranscription.Specific activator proteins, such as CAP in the lac operon,also control transcription of some but not all bacterial genes.These activators bind to DNA together with the RNA polymerase, stimulating transcription from a specific promoter.The DNA-binding activity of an activator is modulated in response to cellular needs by the binding of specific small molecules (e.g., cAMP) that alter the conformation of theactivator.Transcription by 54-RNA PolymeraseIs Controlled by Activators That Bind Farfrom the PromoterMost E. coli promoters interact with 70-RNA polymerase,the major form of the bacterial enzyme.
Transcription of certain groups of genes, however, is carried out by E. coli RNApolymerases containing one of several alternative sigma factors that recognize different consensus promoter sequencesthan 70 does. All but one of these are related to 70 in sequence. Transcription initiation by RNA polymerases containing these 70-like factors is regulated by repressors andactivators that bind to DNA near the region where the polymerase binds, similar to initiation by 70-RNA polymeraseitself.The sequence of one E.
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