Molecular Cell, Vol. 3, 229–238, February, 1999, Copyright 1999 by Cell PressCore RNA Polymerase from E. coliInduces a Major Changein the Domain Arrangement of the s70Subunitthe core enzyme acts as an allosteric modulator of s70DNA binding activity. This model suggests that bindingofs70tothecoreshouldproducealargescalemovementof s70domains.Conformational changes of s70induced by interactionSandhya Callaci, Ewa Heyduk, and Tomasz Heyduk*Edward A. Doisy Department of Biochemistry andMolecular BiologySt. Louis University Medical SchoolSt. Louis, Missouri 63104with the core enzyme were first observed by Wu et al.(1976). Morerecently, we have shown that conformationof conserved domains of s70that were suggested toSummaryparticipate in core-induced structural change (regions1, 2.4, and 4.2) was indeed affected by the core enzymeLuminescence resonance energy transfer measure-(Callaci et al., 1998). Also, the core enzyme inducedments were used to show that binding of E. coli coreconformational changes in s70that could be detectedRNA polymerase induced major changes in interdo-in the region of the protein responsible for binding themain distances in the s70subunit. The simplest modelnontemplate single-stranded DNA (Callaci and Heyduk,describing core-induced changes in s70involves a1998).movement of the conserved region 1 by z20 A˚andIn this work, we investigated the nature of core-the conserved region 4.2 by z15 A˚with respect toinduced conformational changes in s70and addressedconserved region 2. The core-induced movement ofdirectly the question of whether the core enzyme doesregion 1 (autoinhibition domain) and region 4.2 (DNA-indeed induce a large scale movement of s70domainsbinding domain) provides structural rationale for allo-as predicted bythe model(Dombroski etal., 1993b). Thesteric regulation of s70DNA binding properties by theapproach taken was to introduce luminescence donorscore and suggests that this regulation may not onlyand acceptors to different conserved domains of theinvolve directly the autoinhibition domain of s70butprotein and to use luminescence resonance energyalso could involve a modulation of spacing betweentransfer measurements (LRET) (Selvin and Hearst, 1994;DNA-bindingdomains ofs70inducedby bindingofcoreSelvin et al., 1994; Selvin, 1996) to monitor distancesRNAP.between these domains in free s70and in s70-core en-zyme complex. We observed a major rearrangementIntroductionof s70domains consistent with the proposed model.In addition, it was discovered that as a result of coreRNA polymerase from Escherichia coli is a multisubunitpolymerasebinding, DNA-binding domainsofs70movedenzyme composed of two a subunits, large b and b9away from each other such that their spatial separationsubunits, and the s subunit. In the cell, two major formsin the holoenzyme became much more compatible withofthe polymerase arefound: core enzyme(subunitcom-z17 bp separation of 210 and 235 elements of pro-position a2bb9) and holoenzyme (subunit compositionmoter DNA. We propose that this movement of DNA-a2bb9s)(Burgessetal.,1969;BurgessandTravers,1970;binding domains of s70could be an important mecha-Record et al., 1996). The core polymerase is capable ofnism by which the ability of s70to recognize promotertranscription elongation but is unable to initiate tran-DNA is regulated.scriptionatspecificpromotersites.Theinitiation oftran-scription at specific promoter sites is carried out by theholoenzyme (Burgess et al., 1969; Hinkle and Chamber-Resultslin, 1972; Record et al., 1996). Therefore, s subunit isresponsible for the promoter recognition by E. coli RNADonor-Acceptor-Labeled Double-Cysteinepolymerase. However, free s70is not able to bind specifi-Mutants of s70callyatpromoter DNA sites (WellmanandMeares,1991).The overall design of our experiments was to incorpo-This was shown by studies with polypeptide fragmentsrate luminescence donors and fluorescence acceptorss70to be due to the autoinhibition of s70DNA bindingintospecificlocationsinthes70andtousetheresonanceactivitybythe N-terminal domainofs70(Dombroskiet al.,energytransferbetweendonorandacceptortomeasure1992, 1993a). A model of regulation of s70DNA bindinginterdomain distances in free s70and s70bound to theactivity by the core enzyme was proposed based oncore enzyme. Incorporation of a donor into one specificthese data. In this model, the N-terminal domain of s70domain of the protein and the acceptor into some otherwas proposed to be located in the free protein such thatdomain of the same protein is a difficult problem. Weit would sterically block the access of promoter DNA toused Eu31chelate, (Eu31)DTPA-AMCA-maleimide (Hey-DNA-binding domains of s70(regions 2.4 and 4.2). Bind-duk and Heyduk, 1997, 1998), as a donor since theing of s70to the core enzyme was proposed to induceunique luminescence properties of europium chelatesa movement of the N-terminal domain to “unmask” s70greatly facilitate observation and determination of en-DNA-binding domains allowing the protein to recognizeergy transfer in systems where stoichiometric labelingpromoter DNA (Dombroski et al., 1993b). In this model,with donor and acceptor is not possible (Selvin andHearst, 1994; Selvin et al., 1994; Selvin, 1996; Heydukand Heyduk, 1997). We engineered, using site-directed*To whom correspondence should be addressed (e-mail: [email protected]).mutagenesis, pairs of unique reactive cysteine residuesMolecular Cell230and 6 is eliminated by time gating, that is, nanosecondfluorescence of directly excited acceptors decays tozero shortly after the excitation pulseand does not con-tribute to emission-measured microseconds after theexcitation pulse (Selvin and Hearst, 1994; Selvin et al.,1994; Selvin, 1996; Heyduk and Heyduk, 1997). Also,emission of directly excited acceptors in products 1and 2 is eliminated in the same way. Thus, the decayobserved at 617 nm (donor decay) should be a sum ofa decay of unquenched donor (no LRET, products 4, 7,and 8) and a decay of the donor engaged in LRET (prod-ucts 1 and 2). In addition, any emission of the acceptorobserved in a microsecond time range could be onlyduetoexcitationoftheacceptorthroughenergytransferfrom the donor and should decay with the lifetime(s) ofa donor engaged in energy transfer with the acceptor(Selvinand Hearst, 1994;Selvin et al.,1994; Selvin, 1996;Heyduk and Heyduk, 1997). Thus, fitting simultaneouslydecaysofdonor andsensitized acceptor,decay compo-nents due to energy transfer can be