H. Lodish - Molecular Cell Biology (5ed, Freeman, 2003) (796244), страница 49
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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. Note that NtrB and NtrC, discussed above, function as sensor and response regulator proteins, respectively,in the two-component regulatory system that controls transcription of glnA. Similar two-component histidyl-aspartylphosphorelay regulatory systems are also found in plants.KEY CONCEPTS OF SECTION 4.3Control of Gene Expression in ProkaryotesGene expression in both prokaryotes and eukaryotes isregulated primarily by mechanisms that control the initiation of transcription.■Binding of the subunit in an RNA polymerase to apromoter region is the first step in the initiation of transcription in E.
coli.■The nucleotide sequence of a promoter determines itsstrength, that is, how frequently different RNA polymerasemolecules can bind and initiate transcription per minute.■Repressors are proteins that bind to operator sequences,which overlap or lie adjacent to promoters. Binding of arepressor to an operator inhibits transcription initiation.■The DNA-binding activity of most bacterial repressorsis modulated by small effector molecules (inducers).
Thisallows bacterial cells to regulate transcription of specificgenes in response to changes in the concentration of various nutrients in the environment.■The lac operon and some other bacterial genes also areregulated by activator proteins that bind next to promoters and increase the rate of transcription initiation by RNApolymerase.■The major sigma factor in E. coli is 70, but several otherless abundant sigma factors are also found, each recognizing different consensus promoter sequences.■Transcription initiation by all E.
coli RNA polymerases,except those containing 54, can be regulated by repressors and activators that bind near the transcription startsite (see Figure 4-16).■Genes transcribed by 54–RNA polymerase are regulatedby activators that bind to enhancers located ≈100 base■1194.4 • The Three Roles of RNA in Translationpairs upstream from the start site. When the activator and54–RNA polymerase interact, the DNA between theirbinding sites forms a loop (see Figure 4-17).aa 1 aa 2aa 3Growingaa 4polypeptidechainIn two-component regulatory systems, one protein actsas a sensor, monitoring the level of nutrients or other components in the environment.
Under appropriate conditions,the -phosphate of an ATP is transferred first to a histidine in the sensor protein and then to an aspartic acid ina second protein, the response regulator. The phosphorylated response regulator then binds to DNA regulatory sequences, thereby stimulating or repressing transcription ofspecific genes (see Figure 4-18).■H CRibosomeAlthough DNA stores the information for protein synthesisand mRNA conveys the instructions encoded in DNA, mostbiological activities are carried out by proteins. As we sawin Chapter 3, the linear order of amino acids in each proteindetermines its three-dimensional structure and activity.
Forthis reason, assembly of amino acids in their correct order,as encoded in DNA, is critical to production of functionalproteins and hence the proper functioning of cells andorganisms.Translation is the whole process by which the nucleotidesequence of an mRNA is used to order and to join the aminoacids in a polypeptide chain (see Figure 4-1, step 3). In eukaryotic cells, protein synthesis occurs in the cytoplasm,where three types of RNA molecules come together to perform different but cooperative functions (Figure 4-19):1. Messenger RNA (mRNA) carries the genetic informationtranscribed from DNA in the form of a series of threenucleotide sequences, called codons, each of which specifiesa particular amino acid.2.
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