Article No. mb981958 J. Mol. Biol. (1998) 281, 465±473

Visualization of the Binding Site for the TranscriptCleavage Factor GreB on Escherichia coliRNA Polymerase

Andrey Polyakov1, Catherine Richter1, Arun Malhotra1, Dmitry Koulich2

Sergei Borukhov2 and Seth A. Darst1*

1Laboratory of MolecularBiophysics, The RockefellerUniversity, 1230 York AvenueNew York, NY 10021, USA2Department of Microbiologyand ImmunologyState University of New YorkHealth Science Center atBrooklyn, Brooklyn, NY 11203USA

Present address: A. Malhotra, AsDepartment of Biochemistry & MoUniversity of Miami School of Med016129, Miami, FL 33101, USA.

Abbreviations used: 2D, two-dimdimensional; CTD, C-terminal domdomain; RNAP, RNA polymerase,dodecyl sulfate-polyacrylamide gelcircular dichroism.

E-mail address of the corresponddarst@rockvax.rockefeller.edu

0022±2836/98/330465±09 $30.00/0

The structure of Escherichia coli core RNA polymerase (RNAP) complexedwith the transcript cleavage factor GreB was determined from electronmicrographs of negatively stained, ¯attened helical crystals. A bindingassay was developed to establish that GreB was incorporated into theRNA polymerase crystals with high occupancy through interactionsbetween the globular C-terminal domain and the RNA polymerase. Com-parison of the core RNAP:GreB structure with the previously determinedstructure of core RNAP located the GreB binding site on one face of theRNA polymerase, next to but not in the 25 AÊ -diameter channel of RNApolymerase.

# 1998 Academic Press

Keywords: electron microscopy; E. coli RNA polymerase; GreB;transcription elongation factor; transcript cleavage factor

*Corresponding author

Introduction

During each phase of transcription, RNA poly-merase (RNAP) activity is modulated by inter-actions with a wide variety of regulatory factors.Among these are transcription elongation factors,which stimulate the activity of RNAP by increasingthe overall elongation rate. A variety of elongationfactors that regulate RNA synthesis in prokaryotesand eukaryotes have been identi®ed (reviewed byUptain et al., 1997). An appreciation of the import-ance of RNAP regulation during elongation hasbeen stimulated by recent ®ndings implicatinghuman transcription elongation factors in oncogen-esis (Aso et al., 1995; Duan et al., 1995; Shilatifardet al., 1996).

RNA polymerases do not elongate the transcriptat a uniform rate. The RNAP pauses at certain siteson the DNA before resuming elongation, and a

sistant Professor,lecular Biology,icine, P.O. Box

ensional; 3D, three-ain; NTD, N-terminalSDS-PAGE, sodiumelectrophoresis; CD,

ing author:

fraction of the RNAP molecules can becometrapped at these sites, resulting in arrested com-plexes that can neither propagate nor dissociate(Levin & Chamberlin, 1987; Kassavetis &Geiduschek, 1993). One group of transcriptionelongation factors, which so far includes prokaryo-tic GreA and GreB, and eukaryotic SII (TFIIS),increases the overall rate of elongation by greatlymitigating pausing and freeing the complexes fromthe arrested state (Reines et al., 1989; Sluder et al.,1989; SivaRaman et al., 1990; Borukhov et al., 1993).In addition, GreA and GreB can facilitate the tran-sition of RNAP from the stage of abortive initiationto elongation at certain promoters (Hsu et al., 1995)and may also play a role in transcription ®delity(Erie et al., 1993).

GreA, GreB, and SII act by inducing the hydro-lytic cleavage of the nascent transcript within theelongating RNAP (Surratt et al., 1991; Borukhovet al., 1992, 1993; Izban & Luse, 1992; Reines, 1992).Fragments of 2 to 17 nucleotides are cleaved andreleased from the 30-end of the transcript. The 50-terminal fragment of the transcript, bearing a free30-OH, remains bound in the complex and can thenbe extended by RNAP upon the addition ofnucleotides. Reverse translocation of the RNAPalong the DNA template accompanies the cleavagereaction (Feng et al., 1994). The transcript cleavagefactors do not interact with or modify free RNA ontheir own; the transcript cleavage reaction occurs

# 1998 Academic Press

Figure 1. Helical crystals of the E. coli core RNAP:GreBcomplex. (Left) Low magni®cation electron micrographof tubular crystals of E. coli core RNAP with GreB. Thetubular crystals were adsorbed to a glow-dischargedcarbon ®lm and stained with 1% uranyl acetate. (Right)High magni®cation electron micrograph of a ¯attened,tubular crystal of E. coli core RNAP with GreB, stainedwith uranyl acetate.

466 Visualization of GreB on RNA Polymerase

only in the context of the ternary complex withRNAP. This and other observations suggest thatthe RNA cleavage activity is an intrinsic propertyof the RNAP that is induced by the factors (Orlovaet al., 1995).

Escherichia coli GreA and GreB are homologousin sequence with each other and have homologs inmany other prokaryotes. While this family ofprokaryotic proteins has no apparent sequenceor structural homology with eukaryotic SII,stimulation of nascent RNA cleavage in ternarytranscription complexes appears to be an evolutio-narily conserved function. Indeed, an open readingframe encoding a protein product highly homolo-gous in sequence with E. coli GreA is found in thegenome sequence of Mycoplasma genitalium, whichhas the smallest known genome of any free-livingorganism (Fraser et al., 1995). The ubiquity of tran-script cleavage, which has also been observed withvaccinia virus RNAP (Hagler & Shuman, 1993) andeukaryotic RNA polymerases I (Schnapp et al.,1996), II (Izban & Luse, 1992), and III (Whitehallet al., 1994) indicates it plays an important role inthe regulation of transcription.

The 2.2 AÊ resolution crystal structure of GreAcomprises an N-terminal domain consisting of anantiparallel a-helical coiled-coil dimer whichextends into solution, and a C-terminal globulardomain (Stebbins et al., 1995). Based on the highsequence similarity, the functional similarities, andthe fact that GreA and GreB have identical CDspectra, homology modeling was used to derive astructural model of GreB (Koulich et al., 1997). Thestructures led to a model of how the Gre-factorsinteract with RNAP (Stebbins et al., 1995; Koulichet al., 1997). Moreover, functional studies have ledto the conclusion that the Gre-factor C-terminaldomain (CTD) is primarily involved in bindingRNAP, while the Gre-factor N-terminal domain(NTD) interacts with the 30-end of the transcriptand participates directly to induce the transcriptcleavage reaction (Koulich et al., 1997, 1998).Nevertheless, structural studies of complexesbetween the Gre-factors and RNAP or transcriptionelongation complexes will be required to fullyaddress the transcript cleavage mechanism.

Earlier we reported the three-dimensional (3D)structure of E. coli core RNAP to a nominal resol-ution of 23 AÊ , determined by electron microscopyand image processing of ¯attened helical crystalsembedded in negative stain (Polyakov et al., 1995).Here we report an extension of this analysis toE. coli core RNAP complexed with GreB. A bindingassay was used to establish that GreB wasincorporated into the RNAP crystals with highoccupancy via interactions between the globularGreB-CTD and the RNAP. The results localize thebinding site for GreB on one face of the 3Dcore RNAP structure, next to but not in the 25 AÊ -diameter channel of RNAP.

Results

Crystallization of the core RNAP/GreB complex

To locate the site of Gre-factor interaction onthe low-resolution structure of E. coli core RNAP,we compared the 3D structures of the coreRNAP:GreB complex with core RNAP, determinedfrom processed electron micrographs of helicalcrystals. We chose GreB to study the interactionsbetween core RNAP and a transcript cleavage fac-tor for two reasons. First, GreB binds RNAP withan af®nity about two orders of magnitude greaterthan GreA. GreB binds RNAP with a dissociationconstant, Kd, estimated to be about 10ÿ7 M, whileGreA binds with a Kd of about 10ÿ5 M (Orlova,1995). Second, GreA and GreB are homologous inprimary structure (Borukhov et al., 1993), tertiarystructure (Koulich et al., 1997), and function, andso are likely to interact with RNAP in the sameway. This is supported by the ®nding that GreAand GreB act competitively (Koulich et al., 1998).

Thus, E. coli core RNAP was incubated withpositively charged lipids under conditions thatgive rise to tubular crystals with helical symmetry(Polyakov et al., 1995) except that a tenfold molarexcess of GreB over RNAP was present. Examin-ation of the samples preserved in negative stainrevealed the presence of tubular crystals (Figure 1).Interestingly, while the core RNAP helical crystalsrange in diameter from about 350 to 800 AÊ ,the tubular RNAP crystals grown in the presenceof GreB were two to three times that diameter, amorphological difference in the crystals likely dueto the incorporation of GreB into the lattice.

Visualization of GreB on RNA Polymerase 467

We developed an assay to test directly whetherGreB was incorporated speci®cally into the crystallattice. In this assay, tubular crystals of core RNAPwere grown in the presence of a tenfold molarexcess of GreB. Next, the crystallization mixturewas centrifuged to pellet all of the crystals, thesupernatant was removed, and the remaining pel-let was washed brie¯y with crystallization sol-ution. Next, the supernatant and pellet (crystals)fractions were analyzed by SDS-polyacrylamidegel electrophoresis (Figure 2). GreB was found inexcess with the other RNAP subunits in the super-natant fraction (Figure 2, lane 4), but an approxi-mately equimolar amount of GreB was alsoassociated with the RNAP in the pellet (Figure 2,lane 3), indicating approximately stoichiometricbinding to the RNAP crystals. As a control forspeci®city, the same experiment was performedwith streptavidin, a protein not known to interactwith RNAP. Despite the presence of a huge excessof streptavidin in the supernatant (Figure 2, lane7), no streptavidin associated with the pellet(Figure 2, lane 6).

Projection structures

These results demonstrating that GreB wasindeed incorporated into the crystals prompted usto examine the crystals more closely. Analysis oflow-dose, untilted images of the ¯attened coreRNAP crystals grown in the presence of GreB andpreserved in negative stain revealed that the two-dimensional (2D) surface lattice of the crystals wasisomorphous with the crystals of core RNAP to theresolution of the analysis (about 20 AÊ ). However,the core RNAP:GreB surface lattice had a higherradius of curvature, giving rise to the larger diam-eter of the tubes. Thus, the morphology of the crys-tals of core RNAP and of core RNAP:GreB wereidentical after ¯attening, allowing a direct compari-son of the densities using Fourier differencemethods. A projection map was calculated fromten averaged images of untilted core RNAP:GreBcrystals (Figure 3), revealing an obvious extra den-

binding to core RNAP within the tubular crystals. Streptavthis assay (lanes 6 and 13, respectively).

sity when compared with the projection map ofthe core RNAP crystals (Figure 3), even in theabsence of a difference Fourier analysis. A differ-ence Fourier analysis yielded a single, 3s differ-ence peak (Figure 3). We interpreted these resultsto indicate that isomorphous crystals of the coreRNAP:GreB complex were obtained, and that the3s difference peak indicated the position of theGreB molecule in projection.

The structure of GreA, obtained by X-ray crystal-lography, comprises two distinct domains, anN-terminal coiled-coil domain, and a C-terminalglobular domain (Stebbins et al., 1995). Severallines of evidence indicate that GreB has a verysimilar structure. (1) GreA and GreB are homolo-gous in primary sequence (35% identity with onlya one amino acid gap; Borukhov et al., 1993).(2) GreA and GreB have very similar functions andact competitively. (3) The ultraviolet CD spectraand melting pro®les of GreA and GreB are essen-tially identical (Koulich et al., 1997). Recent studiessuggest that the two domains of GreA and GreBparticipate in distinct functions. The Gre-CTDappears to be involved in binding RNAP, whilethe Gre-NTD is directly involved in inducing thetranscript cleavage reaction (Stebbins et al., 1995;Koulich et al., 1997, 1998). We tested this using thedirect binding assay with the GreA-NTD andGreA-CTD. We used the GreA domains becausethe expressed GreB domains were insoluble. Theresults con®rmed the earlier suggestion that theGreA-CTD is the RNAP binding domain (Figure 2,lane 9) while the GreA-NTD did not interact withRNAP on its own (Figure 2, lane 13). Both domainscontained signi®cant amounts of secondary struc-ture as judged by CD spectroscopy (Figure 4). Thevalue for [f]222/[f]208 for the GreA-NTD of about1.1 is indicative of a two-stranded coiled-coil (Lauet al., 1984), while the CD spectra of GreA-CTD isindicative of a primarily b-sheet structure, consist-ent in each case with the X-ray crystal structure(Stebbins et al., 1995).

Since the GreA-CTD bound to RNAP within thetubular crystals, we also examined low-dose,

Figure 2. Direct assay for bindingto RNAP in tubular crystals. Tubu-lar crystals of E. coli core RNAPwere grown in the presence of ten-fold molar excesses of the proteinsindicated on top (GreB, streptavi-din, GreA-CTD, or GreA-NTD).The crystals were then pelleted bycentrifugation and washed. Thesupernatant and pellet (crystal)fractions were then analyzed bySDS-PAGE (4% to 15% gradient)and Coomassie blue staining. Thepresence of an approximately equi-molar amount of the extra proteinin the pellet (as with GreB, lane 3,and GreA-CTD, lane 9) indicated

idin and GreA-NTD did not associate with the RNAP in

Figure 3. Average projection maps calculated from ¯attened helical crystals for core RNAP (upper left) and thecore:GreB complex (upper right). The continuous contours denote stain-excluding density (protein). On the bottom,2s (green) and 3s (red) Fourier difference peaks for [(core:GreB)-core] (lower left) and [(core:GreA-CTD)-core] aresuperimposed on the core RNAP projection map.

468 Visualization of GreB on RNA Polymerase

untilted images of the ¯attened core RNAP crystalsgrown in the presence of GreA-CTD and preservedin negative stain. The analysis of the untiltedimages also revealed the presence of an extra den-sity which yielded a 3s difference peak in thesame position as GreB (Figure 3).

Three-dimensional structure

To determine the 3D location of the GreB bind-ing site on core RNAP, electron images of the coreRNAP:GreB crystals tilted with respect to the elec-tron beam were analyzed. A total of 208 micro-graphs of crystals tilted up to 65� to the incidentelectron beam were collected and used for furtherprocessing. The average phase error, based onre®nement of each new measurement within0.0035 AÊ ÿ1 in z* (the direction in reciprocal space

perpendicular to the crystal plane) was 17.8� whenconsidering re¯ections with amplitudes of signal-to-noise greater than 2. No single micrograph hadan average phase error greater than 30�. Smoothcurves were ®t to the combined amplitude andphase data for each of 39 independent lattice linesincluded in the reconstitution. By sampling thesecurves at intervals of 0.00333 AÊ ÿ1 in z*, 236 Fourierterms were collected and used to calculate the 3Dmap. The quality of the core RNAP:GreB crystalswas not as high as the core RNAP crystals. It wasparticularly dif®cult to obtain highly tilted images(>45�) that diffracted well, probably due to dis-order of the crystals out of the crystal plane. Whilethe 3D data set is sampled well out to about 15 AÊ

resolution in the plane of the crystals, we estimatethe nominal resolution of the 3D core:GreB struc-ture to be about 28 AÊ resolution.

Figure 4. Circular dichroism spectra of GreA-NTD,GreA-CTD, the sum of these two spectra, and intactGreA, all at 4�C.

Visualization of GreB on RNA Polymerase 469

The resulting 3D map revealed clearly the coreRNAP molecule as seen earlier from the coreRNAP crystals (Polyakov et al., 1995), as well as anextra connected density attributed to GreB, asobserved in the projection maps (Figure 3). The 3Dlocation of the GreB density is best visualized by adifference analysis (Figure 5). In the difference map(calculated as described in Materials and Methods),a single 3.5s positive difference peak (shown inred in Figure 5a to c) indicates the position of theGreB molecule with respect to the core RNAP.There were no other positive or negative peaks ofthis magnitude in the difference map. The bindingsite of GreB on core RNAP was delineated by asurface where the GreB density intersected withthe core RNAP density (Figure 5d).

Discussion

The low-resolution, 3D structures of the multisu-bunit cellular RNA polymerases E. coli RNAPholoenzyme (Darst et al., 1989), E. coli core RNAP(Polyakov et al., 1995), and yeast RNA polymerasesI (Schultz et al., 1993) and II (Darst et al., 1991), allshare a common, distinctive feature. Each containsa thumb-like projection that surrounds or de®nes agroove or channel about 25 AÊ in diameter, whichis the appropriate dimensions for accomodatingdouble-stranded nucleic acids. The double-stranded DNA template is presumed to passthrough this channel, and electron crystallographicanalysis of crystallized ternary elongation com-plexes of yeast RNAP II indicate that this is so (C.Poglitsch, G. Meredith & R. D. Kornberg, unpub-lished results). The RNAP active center is pre-sumed to be located at the ¯oor of this channel, inkeeping with X-ray crystal structures of single-sub-unit DNA polymerases, which reveal an analogous(but not homologous) channel feature with the

active site at its ¯oor (Doublie et al., 1998; Kieferet al., 1998).

The main result of this study locates the GreBbinding site on core RNAP. GreB binds on one faceof the RNAP, next to but not in the 25 AÊ channel(Figure 5). This places the closest edge of the GreBbinding site on the surface of core RNAP about30 AÊ away from the presumed location of theRNAP active center. This is consistent with exper-iments indicating that GreB bound to RNAP wasnot cleaved by hydroxyl-radicals generated by Fe2�

chelated at the RNAP active center, placing GreBno less than 10 AÊ away from the RNAP active cen-ter (I. Lomakin & S.B., unpublished results). Thisappears inconsistent, however, with earlier ®nd-ings that a site at the distal end of the coiled-coilGre-NTD on both GreA and GreB crosslinkdirectly to the 30-transcript terminus within ternaryelongation complexes (Stebbins et al., 1995; Koulichet al., 1997). This inconsistency could possibly beexplained in two ways.

First, a number of recent results have revealedthe tendency for RNAP in a ternary elongationcomplex to transiently ``slide back'' or ``backtrack'' on the DNA template with concurrentreverse-threading of the RNA transcript throughthe complex (Reeder & Hawley, 1996; Komissarova& Kashlev, 1997a,b; Nudler et al., 1997). Thisallows for the possibility that the transcript 30-endis not always associated with the RNAP active cen-ter. In fact, RNAP can back track 20 or even morenucleotides, extruding the transcript 30-end fromthe complex (Komissarova & Kashlev, 1997a,b).Komissarova & Kashlev (1997b) found that back-tracked complexes were obligate intermediates inthe Gre-factor mediated transcript cleavage reac-tion, suggesting that the Gre-factors interact withthe transcript 30-end not within the RNAP activecenter but after it has been extruded and displacedfrom it.

Second, we have concluded from the results ofthis study along with previous results from otherstudies (Koulich et al., 1998) that the Gre-CTD isthe primary determinant for binding to RNAP. Inaddition, we tentatively propose that the differencedensity observed here and attributed to GreB(Figure 5) arises primarily from the GreB-CTD (seefurther discussion below). The GreB-NTD may notbe engaged with the RNAP and could thus be dis-ordered and invisible to this analysis. Since theGre-NTD comprises an antiparallel coiled-coil ``®n-ger'' that extends about 45 AÊ from the Gre-CTD(Stebbins et al., 1995; Koulich et al., 1997) it has thepotential to reach from the position of the Gre-CTD to the transcript 30 terminus within or nearthe RNAP active center.

We also investigated the interactions of theGreA-NTD or GreA-CTD alone with the coreRNAP crystals. We found that the GreA-CTDbound to core RNAP within the crystals (Figure 2,lane 9) and a difference Fourier analysis in 2Drevealed a difference peak due to the GreA-CTD inthe same position as the difference peak due to

Figure 5. Binding of GreB in relation to the 23 AÊ -resolution 3D structure of E. coli core RNAP (Polyakov et al., 1995).a to c, The 3D structure of a single E. coli core RNAP molecule is shown as a light blue density. A difference map cal-culated in real space by subtracting the core RNAP density from the core RNAP:GreB density is shown in red, con-toured at 3.5s. There were no other positive or negative peaks at this contour level. The xyz-axes indicate theviewing direction. The labels T and C denote the thumb-like feature and the 25 AÊ -diameter channel, respectively, dis-cussed in the text. We attribute the difference density (red) to GreB (probably GreB-CTD, see the text). To obtain theview shown in a, density connecting adjacent RNAP molecules was cut away (white region). d, E. coli core RNAP isshown in light blue (same view as b). The area where the extra density attributed to GreB in the core:GreB structureintersects the core structure is mapped on the surface of the core RNAP (white).

470 Visualization of GreB on RNA Polymerase

GreB itself (Figure 3). The difference peak due tothe GreA-CTD was weaker than the differencepeak due to GreB itself. If the binding of the GreA-CTD to core RNAP within the crystals was satu-rated, this could be attributed to the smaller mol-ecular mass of the GreA-CTD, which is about halfthe molecular mass of GreB. Alternatively, thebinding of GreA-CTD to core RNAP within thecrystals may not be saturated. While we cannotrigorously distinguish between these two possibili-ties, we favor the latter since the crystal bindingstudies were done with the same molar excesses ofGreB and GreA-CTD over core RNAP, and the

binding af®nity of the GreA-CTD for core RNAP istwo orders of magnitude less than GreB. We wereunable to further increase the concentration of theGreA-CTD in these binding studies to completelysaturate the binding because the core RNAP crys-tals became disrupted. We believe this was due tothe acidic nature of the GreA-CTD (pI � 4.1),which would cause it to non-speci®cally bind tothe positively charged lipids at high concen-trations, possibly disrupting the binding of RNAP.In contrast to the GreA-CTD, the GreA-NTD didnot bind to the core RNAP crystals by itself(Figure 2, lane 13). For this reason, we tentatively

Visualization of GreB on RNA Polymerase 471

propose that the extra density seen in the low-res-olution 3D structure of the core RNAP:GreB com-plex (attributed to GreB) indicates the position ofthe globular GreB-CTD bound to RNAP and thatthe coiled-coil GreB-NTD may be disorderedwithin the crystals and thus invisible to this anal-ysis. This is supported by the size of the differencepeak attributed to the GreB-CTD, which can bedescribed roughly as an oblate ellipsoid withapproximate dimensions of 35 AÊ � 30 AÊ � 15 AÊ

(Figure 5a to c), essentially the same size as theGreA-CTD as seen in the X-ray crystal structure(Stebbins et al., 1995). The 3.5s difference peak cor-responds to about 85% of the volume expected forthe GreB-CTD and about 37% of the volumeexpected for GreB. These results are consistentwith several other lines of evidence indicating thatthe Gre-factor C-terminal globular domain func-tions primarily as an RNAP-binding domain, whilethe N-terminal coiled-coil domain is directlyinvolved in inducing the transcript cleavage reac-tion. For instance, the GreA-CTD is an effectivecompetitive inhibitor of Gre-factor mediated tran-script cleavage, while the GreA-NTD is not(Koulich et al., 1998). The GreA-NTD, on the otherhand, has the ability to induce the transcript clea-vage reaction on its own (Koulich et al., 1998), gov-erns the nature of the cleavage reaction (GreA orGreB-like; Koulich et al., 1997), and can be cross-linked directly to the transcript 30-end at a site dis-tant from the GreA-CTD (Stebbins et al., 1995;Koulich et al., 1997).

The study presented here outlines a generalstrategy that can now be used to identify the bind-ing sites of accessory factors that interact withRNAP. Thus, a mixture of core RNAP and anexcess of the accessory factor (in the present case atenfold molar excess of GreB was used) can beincubated with the appropriate lipids and underthe appropriate conditions for the growth of helicalcrystals. The presence of crystals can then beassessed by examination in the electron micro-scope. If crystals are present, the incorporation ofthe accessory factor within the crystals (and itsapproximate stoichiometry) can be tested by thecrystal binding assay shown in Figure 2. If thecrystal binding assay indicates incorporation of theaccessory factor in a reasonable stoichiometrywithin the crystals, then electron microscopy andimage processing can be used to visualize thelocation of the accessory factor on the RNAP. Co-crystallization of RNAP with an interacting acces-sory factor can yield three possible results.

(i) The binding of the accessory factor toRNAP is incompatible with the molecular packingwithin the RNAP crystals and crystals therefore donot form.

(ii) Crystals of RNAP are able to form but theaccessory factor is excluded from the crystals.

(iii) Crystals of RNAP form and the accessoryfactor is incorporated within the crystal lattice.

The ®rst possibility is excluded by the initial elec-tron microscope examination to determine if crys-tals can form in the presence of the accessoryfactor. The second possibility is excluded by therelatively simple and rapid crystal binding assay,preventing the application of the time-consumingelectron microscopy and image processingmethods to crystals that do not contain the acces-sory factor under study.

Materials and Methods

Proteins

E. coli core RNAP, GreB, and GreA-NTD (GreA resi-dues 1 to 75) and GreA-CTD (GreA residues 76 to 158)were prepared as described previously (Polyakov et al.,1995; Borukhov & Goldfarb, 1996; Koulich et al., 1998).

Crystallization

Helical crystals of core RNAP:GreB were formed onpositively charged lipids by a modi®cation of the lipidlayer crystallization method (Kornberg & Darst, 1991) inwhich the proteins were incubated with unilamellar lipo-somes. A lipid mixture (100 ml) containing 1 mg/mloctadecylamine (Sigma) and 9 mg/ml diphytanoyl-phos-phatidylcholine (Avanti Polar Lipids) in HPLC gradechloroform was dried in a small glass vial under anargon stream. The lipids were then resuspended in200 ml of 45 mM Tris-acetate, 90 mM ammonium acetate(pH 7.8), with vigorous mixing. The lipid mixture wasthen passed through 10 to 20 cycles of freezing andthawing by moving the vial in and out of liquid nitrogenand warm water. The lipid mixture was then extrudedback and forth through a polycarbonate membrane(10 mm pore size; Poretics) using a LiposoFast stabilizer(Avestin, Inc.; MacDonald et al., 1990). The volume ofthe lipid mixture was then adjusted so that the total con-centration of lipids was 8 mM. Examination of the lipo-some sample by electron microscopy revealed thepresence of largely unilamellar vesicles with approxi-mately 10 mm diameter. The liposome sample could bestored at 4�C for many months without apparent degra-dation. The protein sample (typically 20 to 50 ml) con-tained core RNAP (1 mM) and GreB (10 mM) in 50 mMTris-acetate, 100 mM ammonium acetate, 5 mM sper-mine-acetate, 5 mM dithiothreitol (pH 7.8). For helicalcrystal growth, liposomes were added to the proteinsample to a ®nal total lipid concentration of 0.3 mM. Thesample was then incubated at 4�C under an argonatmosphere. After 24 to 36 hours, the crystals were con-centrated by centrifugation for ten minutes using thelowest speed of a table-top microcentrifuge, removal ofall but 10 ml of the supernatant, then gentle resuspensionof the pellet in the remaining 10 ml. Droplets (5 ml) werethen pipetted onto freshly carbon-coated electron micro-scope grids that had been glow discharged for two min-utes After blotting excess solution, the grids werewashed with one drop of distilled water and then nega-tively stained with 1% (w/v) uranyl acetate.

For the crystal binding assay, after crystallization at4�C for 24 to 36 hours, the crystals were pelleted by cen-trifugation at the highest speed of a table-top microcen-trifuge for 30 minutes. The supernatant was thencarefully removed and the pellet was gently washedwith 500 ml of crystallization buffer without disturbing

472 Visualization of GreB on RNA Polymerase

the pellet. The supernatant and pellet fractions were thenanalyzed by SDS-PAGE on a 4% to 15% precast gel(Novex) and Coomassie staining.

Electron microscopy, image processing, andstructure calculations

Electron micrographs were recorded using minimaldose procedures at a magni®cation of 34,000� with aCM12 transmission electron microscope (Philips) operat-ing at 100 kV on SO163 ®lm (Eastman Kodak). The ®lmswere developed for ten minutes in D-19 developer (East-man Kodak). Micrographs were selected by optical dif-fractometry and digitized on an autodensitometer(Perkin-Elmer) with a 20 mm aperture and step size, cor-responding to 5.9 AÊ on the image. The data were pro-cessed by computer using standard methods (Amos et al.,1982; Henderson et al., 1986).

The 3D difference map (Figure 5) was calculated usingcommands within the image processing package SPIDER(Frank et al., 1996). The same low-pass Butterworth ®lterwas applied to the 3D densities for both core RNAP andthe core RNAP:GreB complex. The scaling and differenceanalysis between the two densities (core:GreB±core) wasperformed in real space using the SPIDER command``DR DIFF''. The scaling and difference analysis was per-formed inside a 3D mask constructed to include the coreRNAP and core RNAP-GreB complex densities con-toured at 1.3 and 1.5s, respectively. This was necessaryto prevent large differences between the two maps inregions far away from the molecular densities (such asat very high or low z) from dominating the differenceanalysis.

CD Spectrometry

CD spectra for GreA-NTD and GreA-CTD wereobtained as described previously (Koulich et al., 1997).

Acknowledgments

We are grateful to D. Cowburn and S. Cahill for helpwith the CD measurements. A.P. was supported by anNRSA award (GM 17708-01). S.A.D. is a Pew Scholar inthe Biomedical Sciences. This work was supported inpart by grants from the Irma T. Hirschl Trust, the PewFoundation, and the March of Dimes to S.A.D., and fromthe NIH (GM4098) to S.B.

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Edited by R. Ebright

(Received 25 March 1998; received in revised form 26 May 1998; accepted 27 May 1998)