Lodish H. - Molecular Cell Biology (5ed, Freeman, 2003) (794361), страница 50
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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.
coli sigma factor, 54, is distinctlydifferent from that of all the 70-like factors. Transcriptionof genes by RNA polymerases containing 54 is regulated4.3 • Control of Gene Expression in Prokaryotessolely by activators whose binding sites in DNA, referred toas enhancers, generally are located 80–160 base pairs upstream from the start site.
Even when enhancers are movedmore than a kilobase away from a start site, 54-activatorscan activate transcription.The best-characterized 54-activator—the NtrC protein(nitrogen regulatory protein C)—stimulates transcriptionfrom the promoter of the glnA gene. This gene encodes theenzyme glutamine synthetase, which synthesizes the aminoacid glutamine from glutamic acid and ammonia. The 54RNA polymerase binds to the glnA promoter but does notmelt the DNA strands and initiate transcription until it is activated by NtrC, a dimeric protein.
NtrC, in turn, is regulated by a protein kinase called NtrB. In response to lowlevels of glutamine, NtrB phosphorylates dimeric NtrC,which then binds to an enhancer upstream of the glnA pro(a)NtrC54 polymerase117moter. Enhancer-bound phosphorylated NtrC then stimulates the 54-polymerase bound at the promoter to separatethe DNA strands and initiate transcription. Electron microscopy studies have shown that phosphorylated NtrCbound at enhancers and 54-polymerase bound at the promoter directly interact, forming a loop in the DNA betweenthe binding sites (Figure 4-17).
As discussed in Chapter 11,this activation mechanism is somewhat similar to thepredominant mechanism of transcriptional activation ineukaryotes.NtrC has ATPase activity, and ATP hydrolysis is requiredfor activation of bound 54-polymerase by phosphorylatedNtrC. Evidence for this is that mutants with an NtrC defective in ATP hydrolysis are invariably defective in stimulating the 54-polymerase to melt the DNA strands at thetranscription start site. It is postulated that ATP hydrolysissupplies the energy required for melting the DNA strands.In contrast, the 70-polymerase does not require ATP hydrolysis to separate the strands at a start site.Many Bacterial Responses Are Controlledby Two-Component Regulatory Systems(b)NtrC54 polymerase▲ EXPERIMENTAL FIGURE 4-17 DNA looping permitsinteraction of bound NtrC and 54-polymerase.
(a) Electronmicrograph of DNA restriction fragment with phosphorylatedNtrC dimer binding to the enhancer region near one end and54 –RNA polymerase bound to the glnA promoter near the otherend. (b) Electron micrograph of the same fragment preparationshowing NtrC dimers and 54-polymerase binding to each otherwith the intervening DNA forming a loop between them. [FromW. Su et al., 1990, Proc. Nat’l.
Acad. Sci. USA 87:5505; courtesy ofS. Kustu.]As we’ve just seen, control of the E. coli glnA gene dependson two proteins, NtrC and NtrB. Such two-component regulatory systems control many responses of bacteria tochanges in their environment. Another example involves theE. coli proteins PhoR and PhoB, which regulate transcriptionin response to the concentration of free phosphate. PhoR is atransmembrane protein, located in the inner (plasma) membrane, whose periplasmic domain binds phosphate withmoderate affinity and whose cytosolic domain has proteinkinase activity; PhoB is a cytosolic protein.Large protein pores in the E. coli outer membrane allowions to diffuse freely between the external environment andthe periplasmic space.
Consequently, when the phosphateconcentration in the environment falls, it also falls in theperiplasmic space, causing phosphate to dissociate from thePhoR periplasmic domain, as depicted in Figure 4-18. Thiscauses a conformational change in the PhoR cytoplasmic domain that activates its protein kinase activity. The activatedPhoR initially transfers a -phosphate from ATP to a histidine side chain in the PhoR kinase domain itself. The samephosphate is then transferred to a specific aspartic acid sidechain in PhoB, converting PhoB from an inactive to an activetranscriptional activator. Phosphorylated, active PhoB theninduces transcription from several genes that help the cellcope with low phosphate conditions.Many other bacterial responses are regulated by two proteins with homology to PhoR and PhoB. In each of these regulatory systems, one protein, called a sensor, contains atransmitter domain homologous to the PhoR protein kinasedomain.
The transmitter domain of the sensor protein is regulated by a second unique protein domain (e.g., the periplasmic domain of PhoR) that senses environmental changes.The second protein, called a response regulator, contains a118CHAPTER 4 • Basic Molecular Genetic Mechanisms FIGURE 4-18 The PhoR/PhoBtwo-component regulatory system inE.
coli. In response to low phosphateconcentrations in the environment andperiplasmic space, a phosphate iondissociates from the periplasmic domain ofthe inactive sensor protein PhoR. This causesa conformational change that activates aprotein kinase transmitter domain in thecytosolic region of PhoR. The activatedtransmitter domain transfers an ATP phosphate to a conserved histidine in thetransmitter domain. This phosphate is thentransferred to an aspartic acid in the receiverdomain of the response regulator PhoB.Several PhoB proteins can be phosphorylatedby one activated PhoR. PhosphorylatedPhoB proteins then activate transcriptionfrom genes encoding proteins that help thecell to respond to low phosphate, includingphoA, phoS, phoE, and ugpB.PorinPeriplasmic spaceCytoplasmPhoRsensor (inactive)HPPhoRsensor (active)phoAHPPPPAphoSPphoEDPhoB responseregulator (active)ugpBDPhoB responseregulator (inactive)Inner (cytoplasmic) membraneOuter membranereceiver domain homologous to the region of PhoB that isphosphorylated by activated PhoR.
The receiver domain ofthe response regulator is associated with a second domainthat determines the protein’s function. The activity of thissecond functional domain is regulated by phosphorylation ofthe receiver domain. Although all transmitter domains arehomologous (as are receiver domains), the transmitter domain of a specific sensor protein will phosphorylate only specific receiver domains of specific response regulators,allowing specific responses to different environmentalchanges.
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