Article No. mb982166 J. Mol. Biol. (1998) 284, 195±203

Activator-Sigma Interaction: A Hydrophobic SegmentMediates the Interaction of a Sigma Family PromoterRecognition Protein with a Sliding ClampTranscription Activator

Kevin Wong* and E. Peter Geiduschek*

Department of Biology andCenter for Molecular GeneticsUniversity of CaliforniaSan Diego, 9500 Gilman DriveLa Jolla, CA 92093-0634, USA

E-mail addresses of the corresponepg@biomail.ucsd.edu; kwong@biom

Abbreviations used: gp, gene proglutathione-S-transferase.

0022±2836/98/470195±09 $30.00/0

Activation of transcription at bacteriophage T4 late promoters and coup-ling of late transcription to concurrent replication requires a peculiar tran-scriptional activator, the gp45 sliding clamp of the T4 DNA polymerase.In order to activate transcription, the topologically DNA-linked trimericgp45 must interact with two T4-encoded RNA polymerase-binding pro-teins, the gp33 co-activator, and the gp55 late sigma factor. The carboxytermini of gp55 and gp33 share a similar sequence, which has beenshown to be required for response of late transcription to activation bygp45. Alanine-scanning mutagenesis of the C terminus of gp55 showsthat residues within the short hydrophobic sequence L(D/A)FLYE, arenecessary for gp55 to bind to gp45, and to respond maximally to tran-scriptional activation by gp45. When fused to GST, the peptide SLDFLYEsuf®ces for speci®c gp45 binding. Thus, it constitutes the main gp55 epi-tope for gp45 interaction.

# 1998 Academic Press

Keywords: transcriptional regulation; sigma proteins; T4 gp55; slidingclamps; bacteriophage T4 late genes

*Corresponding authors

Introduction

By and large, activators of transcription areDNA-binding proteins that convey their effects onthe transcription machinery through direct protein-protein contacts. Physical attachment of these pro-teins to DNA is important, because it increaseseffective local concentration (Rippe et al., 1995) andmay be absolutely required for assembling certainmulti-effector complexes with the required geome-try (Grosschedl, 1995; Murakami et al., 1997). How-ever, transcriptional activators that do not requiresite-speci®c, physical DNA attachment are alsoknown. One of these is the bacteriophage N4single-stranded DNA-binding protein (SSB), whoseDNA binding-defective mutants retain the abilityto activate transcription through direct contactswith the RNA polymerase (Miller et al., 1997);another is the sliding clamp of the bacteriophage

ding authors:ail.ucsd.edu

tein; GST,

T4 DNA polymerase, gp45, which also serves asthe activator of transcriptional initiation at bac-teriophage T4 late promoters. The T4 sliding clamprequires only a topological con®nement, instead ofphysical attachment, to DNA in order to activatetranscription (Herendeen et al., 1989, 1992).

Bacteriophage T4 late genes are under the con-trol of simple promoters, consisting of an 8 bpTATA box (with the closely adhered to consensussequence TATAAATA) centered 10 bp upstream ofthe start site of transcription. Accurately initiatedT4 late transcription in vitro requires only gp55, thebacteriophage T4 late transcription sigma familyprotein, and Escherichia coli RNA polymerase coreenzyme (Kassavetis et al., 1983). In contrast, acti-vation of T4 late transcription during the multipli-cation cycle of wild-type T4 phage is much morecomplex, requiring, in addition, an RNA polymer-ase-bound co-activator encoded by T4 gene 33(gp33), the gp45 sliding clamp, and concurrentDNA replication (reviewed by Geiduschek et al.,1997). The underlying mechanisms of this replica-tion-connected transcriptional activation have beendissected in an in vitro system in which transcrip-

# 1998 Academic Press

196 Activator-Sigma Interaction

tional activation requires loading of gp45 ontoDNA by its clamp loader, the gp44/62 complex, atsites such as nicks or primer-template junctionsthat have the formal properties of enhancers(Herendeen et al., 1989; Sanders et al., 1995). Oncethe ring-shaped trimeric gp45 is mounted on DNA,it is free to slide by one-dimensional diffusionalong the DNA thread from its loading site to thepromoter, where it forms the T4 late transcriptioninitiation complex in association with a holoen-zyme consisting of the E. coli RNA polymerasecore (E), gp55 and gp33 (Herendeen et al., 1992;Tinker et al., 1994a,b). How and where the T4 lateholoenzyme and gp45 complex originally link upis not yet clear. What is known is that gp55 canbind to the gp45 sliding clamp as it tracks alongDNA (Tinker-Kulberg et al., 1996), implying thatthe sliding clamp may participate in holoenzymeassembly and in searching T4 DNA sequence forlate promoters.

The replication dependence of this transcrip-tional activation is thought to arise from the lim-ited stability of the trimeric gp45 ring, whichreleases from its DNA-encircling state every time itopens or dissociates. The sites (nicks, gaps and pri-mer-template junctions) that are consequentlyrequired to keep re-loading gp45 onto DNA arethemselves transient, since they are continuouslycreated and consumed by recombination-coupledT4 DNA replication (Fu et al., 1996).

The following evidence speci®es that gp45, theactivator of T4 late transcription, interacts withgp55 and gp33, the two T4 phage-encoded sub-units of the T4 late RNA polymerase holoenzyme:(a) gp45, 55 and 33 lie in proximity to each other atthe upstream end of the promoter complex (Tinkeret al., 1994a); (b) gp45 can bind to gp55 indepen-dently of RNA polymerase as it transits alongDNA (Tinker-Kulberg et al., 1996); (c) gp33 andgp55 can bind directly to gp45 in the absence ofDNA through their C termini (Sanders et al., 1997);(d) these C termini are essential for the activities ofgp33 and gp55 in enhancer-driven, gp45-activatedlate transcription (Winkelman et al., 1994; Sanderset al., 1997). Indeed, three ligands of the gp45 slid-ing clamp: gp55, gp33 and gp43, the T4 phageDNA polymerase; have nearly identical C-terminalamino acid sequences (Figure 1) as previouslypointed out (Berdis et al., 1996; Sanders et al., 1997).

Figure 1. The carboxy termini of gp55, the T4 latetranscription sigma factor, gp33, the co-activator ofenhanced T4 late transcription, and gp43, the T4 DNApolymerase share a core of identical sequence (Berdiset al., 1996; Sanders et al., 1997).

The experiments that are presented belowfurther explore the gp45-gp55 interaction. We haveconducted an alanine-scanning mutagenesis of theessential C terminus of gp55, testing mutants fortheir ability to interact with gp45 and to conveythe activation signal of gp45 in enhancer-driventranscription. These experiments identify individ-ual gp55 residues that are essential for the gp45interaction, and identify a small hydrophobic epi-tope that suf®ces for speci®c interaction withgp45.

Results

Mutagenesis and transcription

Alanine substitutions were made in the C-term-inal amino acid residues 176±185 of gp55 inorder to identify individual residues that areessential for interaction with the sliding clamp(Figure 2A). Mutant genes were overexpressedin E. coli, and the encoded His-tagged proteinswere puri®ed (under denaturing conditions,as described in Materials and Methods). TheN-terminal His6 tag has no effect on the proper-ties of wild-type gp55 in basal or gp45-activatedin vitro transcription (data not shown; and seeLeÂonetti et al., 1998).

The rates of the formation of open promotercomplexes in basal transcription by E. coli RNApolymerase with wild-type gp55 and each of thesegp55 mutants were compared. If the alanine substi-tutions affected only the interaction with gp45 andgp45-mediated transcriptional activation, then thecorresponding proteins should be unaffected intheir basal rates of initiating transcription at a T4

Figure 2. Determinants of interaction with gp45.A, Effects of point mutations in the C terminus of gp55on ability to direct unenhanced and enhanced transcrip-tion, and to bind gp45. B, GST-gp55-C-end fusion pro-teins that bind speci®cally to gp45. n.d., not determined.

Figure 3. Rates of promoter opening in basal tran-scription. A, The dependence of production of the 420 ntT4 late transcript in a single round of transcription ontime of incubation of linear pDH310 with gp55-contain-ing RNA polymerase. Data for wild-type gp55, gp55-L180A and gp55-F179A are shown. A 305 nt DNArecovery marker was used to quantify relative yields oftranscripts. B and C, Transcript yields in single roundsof transcription as a function of the time of promoteropening for gp55 point mutants are shown relative towild-type gp55 (set to 1.0 at t � 30 minutes). Each plotis the average of two or more experiments.

Activator-Sigma Interaction 197

late promoter. This was examined in experimentsin which gp55 and RNA polymerase core werepreincubated, and added to linear DNA containinga T4 late promoter for the indicated time (Figure 3)before addition of ribonucleoside triphosphatesand rifampicin for a single round of transcription(the yield of transcripts serving as a proportionalmeasure of the formation of open promoter com-

plexes). With the exception of gp55-E182A and-D185A, the alanine-substituted proteins retainedwild-type gp55 levels of activity, implying reten-tion of ability to bind RNA polymerase, recognize,and open the T4 late promoter. Removal of thethree C-terminal residues, A183, N184 and D185(gp55C�3) or amino acid residues 178±185(gp55C�8) also had no effect on basal transcription(Figure 2A; and data not shown; Winkelman et al.,1994).

The possibility that the decrease in activity forunenhanced transcription of the E182A and D185Amutant proteins relative to wild-type gp55 (45 and30%, respectively; Figure 3C) might stem from atendency to aggregate during renaturation wasexamined by gel-exclusion chromatography onSuperose 12. When gp55-E182A was renatured by100-fold dilution of its 6 M guanidine storage buf-fer, it became highly aggregated, with most of theprotein eluting in the excluded volume. Gp55-D185A behaved similarly, a large proportion elut-ing in the excluded volume or as a dimer (data notshown). The observation that extensive aggrega-tion of these two mutant proteins had only a mod-est effect on transcriptional activity is in contrast towhat was found when charged-to-hydrophobicamino acid substitutions were introduced into agp55 domain that had been implicated in bindingto RNA polymerase core (LeÂonetti et al., 1998). Inthat case, a gp55 mutation generating a highdegree of aggregation also inactivated basal T4late transcription. It appears that the observedaggregation of gp55-E182A and -D185A does notgrossly interfere with their RNA polymerase coreinteraction. Indeed, RNA polymerase binding andpromoter complex formation may partially pre-vent the aggregation of these mutant proteins,perhaps by competing for a site of potential self-association.

Alanine substitution proteins were tested nextfor response to activation by gp45 in enhancedtranscription. In these experiments, RNA polymer-ase core, wild-type or mutant gp55, gp45, gp44/62complex, and gp33, were pre-incubated beforeaddition of the enhancer, and T4 late promoter-containing DNA together with gp32 (the T4 single-stranded DNA-binding protein) and dATP for theindicated time (Figure 4) before initiating a singleround of transcription. Substituting alanine forresidue L177, F179, L180, Y181 or E182 caused asigni®cant loss of activation by gp45 (Figure 4), butit was surprising to see that substitution for theconserved D178 did not affect transcriptional acti-vation by gp45 (Figure 4B). Alanine substitutionfor N184 and D185 generated intermediate effects.For gp55-N184A, the initial rate of promoter open-ing was barely affected, but the maximum level oftranscription was approximately 30% lower thanfor wild-type gp55 (average of four determi-nations). When corrected for this discrepancy, pro-moter opening rates for gp55-N184A and for thewild-type protein were not signi®cantly different.For gp55-D185A, the rate of promoter opening in

198 Activator-Sigma Interaction

enhanced transcription was lower than for wild-type gp55, even when allowance was made for a30% lower activity in unenhanced, basal transcrip-tion (Figure 3C). Nevertheless, gp55-D185A wasseen to have a higher level of activity in enhancedtranscription than the group of alanine substitutionmutants comprising L177A, F178A, L180A and

Figure 4. Rates of promoter opening in sliding clamp-activated transcription. A, The dependence of pro-duction of the 420 nt T4 late transcript in a single roundof transcription on the time of promoter opening isshown (as in Figure 3A). The linear pDH310 templatehas a site for loading gp45 with appropriate polarity(Herendeen et al., 1989; Sanders et al., 1995) for tran-scriptional activation. Data for wild-type gp55, gp55-L180A, and gp55-F179A are shown. B and C, Transcriptyields in single rounds of enhanced transcription as afunction of the time of promoter opening for gp55 pointmutants, relative to wild-type gp55 (set to 1.0 at t � 15minutes). Each plot is the average of two or more exper-iments. Error bars indicate the standard error of themean. Symbols have the same meaning as in Figure 3.

Y181A, and a somewhat higher level of activitythan gp55-E182A. These properties of gp55-D185Awere put in perspective by noting that wild-typegp55 and gp55C�3 are indistinguishable in theirrates of late promoter opening in gp45-activatedtranscription (data not shown).

gp45 binding

The possibility that the gp55 mutations speci®-cally affecting enhanced transcription also generatea loss of interaction with gp45 was explored bygp45-gp55 af®nity chromatography. gp55 wasimmobilized on Ni-NTA agarose through itsN-terminal His6 tag, columns were washed toremove unbound gp55, and gp45 was thenallowed to bind (as described in Materials andMethods). After washing with 0.5 M and 1.0 MNaCl, bound proteins were eluted with SDS andEDTA (Sanders et al., 1997). The results of the anal-ysis correlated strongly with the outcome of theenhanced transcription experiments. Wild-typegp55 bound gp45 tightly, with no gp45 releaseduntil gp55 itself was eluted with SDS and EDTA(Figure 5A and B). gp55C�8 (lacking amino acidresidues 178±185), which was previously shownnot to bind gp45 (Sanders et al., 1997) served as anegative control (Figure 5A). The gp55 alanine sub-stitution proteins that were de®cient in gp45-acti-vated transcription, -L177A, -F179A, -L180A,-Y181A and -E182A, behaved like gp55C�8 anddid not bind gp45. Mutant proteins that retainedgp45 activation, gp55-D178A and -N184A, retainedtheir ability to bind gp45, and so did the partlyactivation-de®cient gp55-D185A (Figure 5B anddata not shown). In the absence of gp55, gp45 didnot bind to Ni-NTA agarose.

The C terminus of gp55 suffices forgp45 binding

In order to specify the minimum epitope of gp55suf®cing for speci®c binding to the gp45 slidingclamp, we constructed a series of genes fusinggp55 C-terminal peptides to the C terminus ofglutathione-S-transferase (GST) through a short,hydrophilic and ¯exible linker (RGSGGGSS;Figure 2B). The encoded proteins were overpro-duced, puri®ed and tested for their ability to bindgp45 by af®nity chromatography (Figure 6). EachGST-fusion protein was immobilized on gluta-thione-Sepharose, and gp45 was adsorbed in bufferwith 100 mM NaCl. The column was washed withthe same buffer, and GST-fusion protein and anymaterial remaining bound to it were then elutedwith SDS and EDTA. Because of the similar sizesof the GST-fusion proteins and gp45, the latter wasdetected by immunoblotting (see Materials andMethods).

GST-55C�3, which contains the amino acid resi-dues shown to be individually essential for inter-

Figure 5. Af®nity chromatography of gp45 on immobilized wild-type and mutant gp55. N-terminally His-taggedgp55 wild-type, gp55-C�8, -F179A, -L180A and -Y181A (A), as well as gp55-E182A, -N184A and -D185A (B) wereseparately bound to Ni-NTA agarose and gp45 was applied. A ¯ow-through-wash fraction (FT) was collected, andthe column washed successively with 0.5 M NaCl and 1.0 M NaCl, before eluting remaining protein with SDS/EDTA(E). An equi-proportional aliquot of the applied gp45 is shown in the left-hand lane (L). Proteins are identi®ed at theleft.

Figure 6. Af®nity chromatography of gp45 onimmobilized GST-fusion proteins. GST-55C�8, GST-D(178)A, GST-L(180)A, and GST-55C were bound toglutathione Sepharose. gp45 was applied in buffer ACwith 100 mM NaCl (load in lane L), the ¯ow-through(FT) was collected, the column was washed twice (W1and W2), and bound protein was eluted with SDS/EDTA (E). gp45 was detected by immunoblotting.

Activator-Sigma Interaction 199

action with gp45 (Figure 5), was seen to bind gp45at protein concentrations similar to those used forthe gp45-af®nity chromatography of intact gp55(Figure 6). GST-55D(178)A, which served as a posi-tive control, since full-length gp55 with the D178Asubstitution was seen to bind to gp45 (Figure 5A),and GST-55L(180)A, which served as a negativecontrol in view of the failure of gp55-L180A tobind gp45 (Figure 5A), gave consistent results:GST-55D(178)A did, and GST-55L(180)A did not,bind gp45 (Figure 6). GST alone, and glutathione-Sepharose also did not bind gp45 (data notshown). The large quantities of GST eluting fromthe column with gp45 were found not to interferewith recognition of gp45 by its antibody.

Discussion

These results suggest that the sequence L(D/A)FLYE at the C terminus of gp55 is a minimalepitope that suf®ces for binding to the gp45 slidingclamp. The hydrophobic nature of this epitopeimplies that its target in gp45 is a hydrophobic sur-face patch or near-surface pocket. There must be atleast some tolerance for variation at the twoC-proximal positions of this sequence: although

200 Activator-Sigma Interaction

mutation of Y181 to alanine disrupts gp45 binding,the presence of Leu and Phe at the correspondingpositions in the C termini of gp33 and gp43,respectively (Figure 1), indicates that these otherhydrophobic amino acid residues are compatiblewith gp45 binding. Changing E182 in gp55 to Adisrupts gp45 binding, while gp43 has G at the cor-responding position and gp33 lacks this residuealtogether. These differences may be at the originof the observed differences of af®nity for gp45:gp43 binds only weakly to gp45 in the absence ofDNA (Latham et al., 1997b) and gp55 is suggestedto bind more tightly to gp45 than does gp33(Sanders et al., 1997).

The postulated hydrophobic domain on the sur-face of gp45 may also interact with other T4 pro-teins that lack this exact peptide sequence (that is,L(D/A)FLYE). It has been reported that the inter-action of the gp44/62 clamp-loading complex withgp45 can be competitively blocked by the octapep-tide (NH3

�)CSLDFLFG(COOÿ) (Berdis et al., 1996).A potential candidate sequence, HLAYLFI, at theC terminus of gp44 might be suitable for a hydro-phobic interaction with gp45, but that remains tobe determined. Deletion of the C-terminal 20amino acid residues of gp41, the T4 DNA helicase,also disrupts an interaction with the T4 DNA poly-merase accessory proteins (Richardson & Nossal,1989). There is no such candidate (contiguous)hydrophobic sequence within this segment,suggesting that the C-terminal segment of gp41does not interact directly with gp45, or that it con-tacts a different site on gp45 from that used bygp55. The relatively rare occurrence of LDFL(Y/L/F) at or near the C termini of proteins and its com-plete absence from the C termini of E. coli proteins(R. Doolittle, personal communication) suggeststhe existence of a niche for speci®c and distinctiveinteraction that has been exploited for interactionswith the versatile and multi-functional gp45 slidingclamp. Indeed, one can think of this interactionas lying at the heart of a regulatory systemthat couples selective gene activation to DNAreplication.

Nevertheless, ®nding that this short C-terminalpeptide is suf®cient for binding to gp45 does notrule out the possibility that other regions of gp55are involved in interactions with gp45. In fact,although removal of the C termini of both gp55and gp33 drastically reduces the level of transcrip-tion (Sanders et al., 1997), a very weak residualactivation can still be observed, consistent with theexistence of additional sites of gp45-interaction ingp55, gp33, or one of the RNA polymerase coresubunits. That gp45 binds less tightly to the C tailof gp55 than to intact gp55 is also suggested bydirectly comparing Figures 5 and 6, and by theobservation that binding of gp45 to the GST fusionproteins is more sensitive to elevated salt than isbinding to gp55 (data not shown). Since gp45 is atrimer it is, in principle, capable of binding gp55,gp33, and even a third ligand, concurrently. Thefact that the elimination of just one of these inter-

actions drastically reduces gp45-mediated tran-scriptional activation suggests that the latter is notsolely generated by polymerase recruitment to thepromoter.

The interaction between gp55 and gp45resembles other prokaryotic RNA polymerase-acti-vator interactions in two respects. First, the smallsize and the very C-proximal positioning of theessential epitope of gp55 is reminiscent of the inter-action domains of both the phage f29 transcrip-tional activator p4 and the phage N4 SSB protein:the C-terminal R120 of p4 is essential for activatingtranscription in Bacillus subtilis (Mencia et al., 1996),and N4 SSB requires its ®ve C-terminal amino acidresidues to activate transcription through contactswith the E. coli RNA polymerase b0 subunit (Milleret al., 1997). Second, activation of transcriptionthrough interactions with the C-terminal portionsof prokaryotic RNA polymerase subunits is fairlycommon. The C termini of the a (Mencia et al.,1996), s (Li et al., 1994) and b' (Miller et al., 1997)subunits of RNA polymerase have all been ident-i®ed as speci®c targets of transcriptional activators.It is interesting to note that a number of viral DNApolymerases, including the HSV1 and pseudo-rabies enzymes, utilize hydrophobic interactionsof C-terminal domains with accessory proteinsto increase processivity of DNA synthesis(Berthomme et al., 1995; Digard et al., 1993;Hernandez & Lehman, 1990; Stow, 1993).

It has recently been shown that the same faceof gp45 (the face where the C termini of its con-stituent monomers lie) is involved in interactionswith both gp43 and the gp44/62 complex(Latham et al., 1997a,b). The implication of our®ndings and of previous work (Sanders et al.,1995) is that this face of gp45 also interacts withgp55 and gp33. Examination of the recentlydetermined structure of gp45 (unpublished; gen-erously provided by J. Kuriyan) identi®es a can-didate hydrophobic-and-basic surface patch onthe C-terminal face of gp45 trimer for potentialinteraction with the hydrophobic-and-acidic tailof gp55. Of course, the actual site of interactionremains to be determined.

Materials and Methods

Mutagenesis, protein production andprotein purification

Mutations were introduced into the C terminus ofgp55 by polymerase chain reaction (PCR) using muta-genic oligonucleotide primers and Pfu DNA polymerase.(Sequences of primers and plasmids are available uponrequest.) The template for PCR was pPLHG55, a pre-viously described over-production plasmid for gp55(Williams et al., 1987). PCR products were insertedbetween the NcoI and BamHI sites of a pET21b vectorderivative (from A. Kumar), introducing six histidineresidues between the ®rst and second residues of gp55.These genes were expressed in E. coli BL21lDE3, grownat 37�C to A600 � 0.6±1.0, and induced with 0.5 mM iso-

Activator-Sigma Interaction 201

propyl-b-D-thiogalactopyranoside (IPTG) for two to threehours. Wild-type and mutant gp55 were puri®ed frominclusion bodies essentially as described (LeÂonetti et al.,1998), dialyzed into storage buffer (6 M guanidiniumchloride, 5% (v/v) glycerol, 200 mM NaCl, 2 mM b-mer-captoethanol, 33 mM KHepes (pH 7.8)), and stored atÿ70�C.

Genes encoding GST fusion proteins were constructedutilizing the pGEX2TK plasmid. Complementary oligo-nucleotides encoding the peptides RGSGGGSSLDFLYE(for GST-55C�3), RGSGGGSSLDFLYEAND (GST-55C),RGSGGGSSLAFLYE (GST-55D(178)A) and RGSGGGSS-LDFAYE (GST-55L(180)A) were annealed and insertedbetween the BamHI and EcoRI sites of pGEX2T. Correctinsertions were veri®ed by restriction analysis and DNAsequencing. These GST fusion protein-encoding geneswere expressed in E. coli DH5a, grown at 37�C toA600 � 0.6±1.0, and induced by adding IPTG to 0.5 mMfor two to three hours. The collected cells, resuspendedin PBS (0.14 M NaCl, 3 mM KCl, 12 mM(P) Na/K phos-phate (pH 7.3)), were lysed by sonication. The clearedlysates were loaded onto glutathione-Sepharose 4B col-umns, pre-equilibrated with PBS, and the columns werewashed three times with ®ve volumes of PBS. BoundGST-fusion proteins were eluted with a buffer containing50 mM Tris-HCl (pH 8.0), 10 mM reduced glutathione,120 mM NaCl, dialyzed into storage buffer containing20 mM Hepes (pH 7.8), 10 mM MgCl2, 1 mM Na3EDTA,20% (v/v) glycerol, 5 mM b-mercaptoethanol, andstored at ÿ70�C. Protein concentration was determinedby absorbance (A280 � 1 corresponding to 0.5 mg/mlprotein).

The preparation of gp33, gp32, amino-terminally His-tagged gp55C�8 and full-length gp55, RNA polymerasecore enzyme from uninfected E. coli, gp44/62 complexand gp45 has been reported or referenced previously(Herendeen et al., 1990; Sanders et al., 1997; Williamset al., 1987, 1989).

Single-round transcription

DNA templates for transcription were derived frompDH310 (Herendeen et al., 1989). For basal transcription,pDH310 was linearized by digestion with ScaI. Forenhanced transcription, pDH310 was linearized, treatedwith ExoIII and puri®ed as described (Sanders et al.,1995).

For basal transcription, 36 pmol of N-His-tagged wild-type or mutant gp55 was incubated on ice with 6 pmolof RNA polymerase in 40 ml of transcription buffer(240 mM potassium acetate, 33 mM KHepes (pH 7.8),10 mM magnesium acetate, 1 mM DTT, 150 mg/mlBSA), transferred to 25�C, and added to 0.75 pmol of lin-ear pDH310 DNA in 40 ml of transcription buffer (pre-equilibrated at 25�C). At speci®ed subsequent times,transcription was initiated by transferring a 10 ml aliquotto 5 ml of NTP mix (5 mM GTP, 5 mM ATP, 0.5 mMCTP, 0.5 mM [a-32P]UTP (4000 cpm/pmol), 125 mg/mlrifampicin in transcription buffer). Transcription wasallowed to proceed for eight minutes and halted by add-ing 150 ml of stop buffer (20 mM Na3EDTA, 40 mM Tris-HCl (pH 8), 250 mM NaCl, 0.4% (v/v) SDS, 250 mg/mlyeast RNA) containing a small quantity of labeled DNAfragment as a sample recovery marker. Transcripts werepuri®ed, resolved and quanti®ed as described (LeÂonettiet al., 1998).

For sliding clamp-activated, enhanced transcription, aDNA mix containing, in 40 ml of transcription buffer,

0.6 pmol of Exo III-treated DNA, 6.1 mg of gp32 and3 mM dATP, and a protein mix containing, in 40 ml oftranscription buffer, 6 pmol of RNA polymerase,30 pmol of gp33, 40 pmol of gp44/62 complex, 22 pmolof gp45 trimer and 36 pmol of mutant or wild-typegp55, were separately equilibrated at 25�C. At time zero,the DNA mix was added to the protein mix, and at theappropriate subsequent times, 10 ml aliquots wereremoved to 5 ml of NTP mix to start a single round oftranscription. Samples were processed as indicatedabove.

Aggregation of gp55 mutants

Wild-type or mutant gp55 (�240 pmol) was diluted100-fold out of storage buffer into 200 ml buffer D � 500(20 mM NaHepes (pH 7.8), 7 mM MgCl2, 500 mM NaCl,10% (v/v) glycerol, 0.006% (w/v) Tween 20, 0.004 mMNa3 EDTA, 10 mM b-mercaptoethanol, 5 mM PMSF, and0.5 mg/ml each of leupeptin and pepstatin) containingmarker proteins (cytochrome c, ovalbumin, bovineserum albumin, aldolase, catalase, and ferritin) for sizecalibration, and loaded onto a Superose 12 column pre-equilibrated with the same buffer. The eluate was ana-lyzed by immunoblotting with rabbit antibody to gp55,as described (LeÂonetti et al., 1998).

Affinity chromatography

Af®nity columns for analyzing gp45-gp55 interactionwere assembled as described (Sanders et al., 1997). Analiquot of gp45 (80 pmol) in 20 ml of buffer A (160 mMpotassium acetate, 20 mM KHepes (pH 7.8), 10 mMmagnesium acetate, 0.05% (w/v) Brij 58, 5% (v/v) gly-cerol and 1 mM b-mercaptoethanol) was allowed to bindto each column, which was subsequently washed with40 ml of buffer A (¯ow-through fraction) followed by60 ml each of buffer W (20 mM KHepes (pH 7.8), 0.05%Brij 58) with 0.5 M and 1.0 M NaCl added, respectively.Bound proteins were eluted with 60 ml of 0.1% SDS/100 mM Na3EDTA. Proteins in each fraction were ana-lyzed by SDS-PAGE.

Glutathione Sepharose was pre-equilibrated in PBSand incubated with 2 mg of GST fusion protein per mlof settled Sepharose, washed extensively in buffer AC(40 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 0.1% Tween20, 10% glycerol, 10 mM b-mercaptoethanol, 100 mMNaCl) and stored at 4�C. Mini-columns of 30 ml (settledvolume) GST-bound Sepharose were washed with 200 mlof buffer AC. Eighty pmol of gp45 in 20 ml AC bufferwas loaded onto each column, which was then washedwith 40 ml of buffer AC (collected as ¯ow-through). Aftertwo further washes (W1 and W2) with 60 ml buffer AC,GST fusion proteins and bound gp45 were eluted with40 ml of 0.1% SDS/100 mM Na3EDTA. Proteins in thesefractions were resolved by SDS-PAGE and detected byimmunoblotting with rabbit antibody to gp45, visualizedwith alkaline phosphatase conjugated to goat anti-rabbitantibody (Harlow & Lane, 1988).

Acknowledgments

We thank G. M. Sanders for guidance and advice atthe outset of these experiments, J. Kuriyan for gener-

202 Activator-Sigma Interaction

ously providing the coordinates of gp45 in advance ofpublication, R. L. Doolittle, G. A. Kassavetis, S. Kolesky,J.-P. LeÂonetti and M. Ouhammouch for helpful discus-sions. We thank J.-P. LeÂonetti and M. Ouhammouch fordetailed and insightful comments on the manuscript.K.W. has been supported by a training grant in CellBiology, Molecular Biology and Genetics from theNIGMS. We are grateful for support of this research by agrant from the NIGMS.

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Edited by R. Ebright

(Received 14 May 1998; received in revised form 29 July 1998; accepted 30 July 1998)