Conformational Changes of Escherichia coli σ54-RNA-Polymerase upon Closed–Promoter Complex Formation

doi:10.1016/j.jmb.2005.09.057

J. Mol. Biol. (2005) 354, 201–205

C OMMUNICATION

Conformational Changes of Escherichia coli s54-RNAPolymerase upon Closed–Promoter Complex Formation Pampa Ray†, Richard J. Hall†, Robert D. Finn, Shaoxia Chen Ardan Patwardhan, Martin Buck* and Marin van Heel* Department of Biological Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ UK

RNA polymerase from the mesophile Escherichia coli exists in two forms, the core enzyme and the holoenzyme. Using cryo-electron microscopy and single-particle analysis, we have obtained the structure of the complete RNA polymerase from E. coli containing the s54 factor within the closedpromoter complex. Comparisons with earlier reconstructions of the core enzyme and the s54 holoenzyme reveal the behaviour of this major variant RNA polymerase in defined functional states. The binding of DNA leads to significant conformational changes in the enzyme’s catalytic subunits, apparently a necessity for the initiation of enhancer-dependent promoterspecific transcription. q 2005 Elsevier Ltd. All rights reserved. 54

*Corresponding authors

Keywords: RNA polymerase; s ; promoter; cryo-EM; single particle analysis

Transcription is a critical step at which the expression of genes responsible for cell adaptation, differentiation and growth is regulated. The multisubunit, DNA-dependent RNA polymerases (RNAPs), are responsible for the transcription of DNA into RNA and are the targets of many regulators of gene expression.1 The best studied prokaryotic RNAP is that from the mesophile Escherichia coli (EC 2.7.7.6) and exists in two forms. The core enzyme (E, subunit composition: a2bb 0 u) is catalytically competent and is required for transcription elongation but is incapable of binding specifically to DNA. For promoter-specific transcription to occur, a s factor binds to the core

† P.R. & R.J.H. contributed equally to this work. Present addresses: P. Ray, Laboratory of Cell Biochemistry and Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA; R. J. Hall, Physical Biosciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA 94720, USA; R. D. Finn, The Wellcome Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambs. CB10 1SA, UK; S. Chen, Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Abbreviations used: RNAP, RNA polymerase; EM, electron microscopy. E-mail addresses of the corresponding authors: [email protected]; [email protected]

enzyme to form a holoenzyme (a2bb 0 us).2 In E. coli, there are two classes of s factors, which have been divided according to differences in their primary sequence and their mode of activation. The s70 class, containing six members, includes factors responsible for the expression of most genes during the exponential growth phase. The s54 factor constitutes a class by itself and is the factor needed for the expression of genes involved in diverse adaptive responses. Although using the same core enzyme, these two classes of s factors are involved in very different types of transcription activation processes.3 The s70 type factor (called sA in some bacteria) has sequence-specific contacts centred at K10 and K35 relative to the C1 transcription start site. In contrast, s54 can bind to promoter DNA alone, and recognises a K24, K12 dinucleotide consensus sequence.4 Activation involving the holoenzyme containing the s70 factor (Es70) is often mediated by increasing promoter occupancy.5 On the other hand, Es54-dependent transcription activation occurs at the DNA melting step and resembles eukaryotic transcription initiation in its requirement for an oligomeric enhancer-binding activator (a AAAC protein) and ATP. The AAAC activators hydrolyse ATP for conversion of the Es54 closed complex to the open promoter complex.6 The aim of this study was to gain structural insights into the functional states of Es54 RNAP. Using transmission electron microscopy and single-particle

0022-2836/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

202

s54-RNA Polymerase Holoenzyme–DNA Complex

(Es54);8 and (iii) a closed-promoter complex with promoter DNA (Es54-DNA). Our studies follow from earlier reconstructions of the core enzyme7 and the s54 holoenzyme.8,9 Three-dimensional structure of the native Es54DNA complex Figure 1. Comparison of (a) and (c) E. coli Es54 DNA cryo-EM structure with (b) and (d) Thermus aquaticus EsADNA crystal structure. The a subunits are indicated in green, b red, b 0 blue, u yellow and s in orange. Grey and black indicate DNA. White (enclosing a black square) indicates the proposed DNA density in the EM structure. The arrow (/) indicates the K10 (downstream) end of DNA in the crystal structure. The final ratio of E to s54 to DNA in the sample used for cryo-electron microscopy was 1:1.5:2, and the concentration of the complex was w0.2 mg/ml. Micrographs were acquired on a Philips CM200 FEG microscope at a magnification of 50,000! using a range of defocus values (w0.9–2 mm underfocus). Eleven micrographs were digitised using a Leafscan 45 densitometer at a step size of 10 mm (corresponding to ˚ on the specimen stage). A semi-automated 2.021 A particle selection methodology was applied to select over 12,000 particles.15 The selected particles were extracted into frames of 128!128 pixels. Contrast transfer function (CTF) correction by phase flipping was applied following the calculation of the precise defocus parameters of individual micrographs up to spatial frequen˚ . All subsequent image processing was cies of 10 A performed using the IMAGIC-5 software package.30 An earlier cryo-EM structure of Es54 was used to generate starting reference projections.8 These references were used for multi-reference alignment (MRA)31 of the initial data set. Multivariate statistical analysis (MSA) and automatic classification were used to generate the initial characteristic class averages. Angular reconstitution32 was used to obtain the relative Euler angle orientations of the selected class averages using the Es54 structure as a starting reference. A 3D reconstruction was then obtained by applying the exact-filtered back-projection algorithm.33 Class averages were then compared with reprojections in corresponding directions generated from the 3D reconstruction, in order to verify the accuracy of the Euler angles assignment. A set of 29 anchor reprojections was used to refine the angular orientation of the set of class averages in each refinement cycle. At the end of each cycle, the 3D reconstruction was masked to remove external noise and reprojected to generate a new large set of references, for further iterations of MRA, MSA, Euler angle assignment and 3D reconstruction.14 The 3D analysis was refined iteratively until a stable 3D reconstruction was obtained. The final resolution of reconstruction was assessed by Fourier shell correlation using the 1⁄2 -bit threshold criterion.33,34 For the final representation of the results, a band-pass filter was applied to the 3D structures to suppress low-frequency ˚ and high frequency comcomponents below 160 A ˚. ponents above 21 A

analysis of frozen-hydrated specimens, we show that the core RNAP, within the transcription cycle, adopts three distinct conformations in solution: (i) a free-core conformation (E);7 (ii) a s54-bound form

The components of Es 54 were purified by conventional chromatographic procedures.10–11 Having established that Es54 formed correctly in vitro by non-denaturing polyacrylamide gel electrophoresis, the reconstituted holoenzyme was incubated with a purified 36 base-pair DNA fragment, called early melted DNA. This sequence, spanning from position K35 to position C1, originates from the nifH promoter from Sinorhizobium meliloti and contains a heteroduplex segment at K11/K12. This heteroduplex segment mimics the conformation of DNA within closedpromoter complexes.12 Earlier footprinting studies have shown that the K10 to K1 segment of the DNA would not be melted in the unactivated complex,13 and that the promoter construct used provides a tight binding site for Es54. A Philips CM200 FEG transmission electron microscope was used to obtain cryo-electron microscope (EM) images of the native Es54KDNA complex. Buffer conditions established for effective closed-promoter complex formation (including salt and glycerol) were preserved at the cost of some loss of contrast in the micrographs. A total of 12,000 molecular images was used for extensive image processing,14,15 resulting in a structure at a final ˚ (Figure 1(a) and (c)). Figure 2 resolution of w24 A illustrates three distinct conformations that RNAP can adopt. A comparison of the new structure (Figures 1(c) and 2(c)) with the cryo-EM structure8 of Es54 (Figure 2(b)), revealed significant differences, notably in the mobile b and b 0 domains. In crystallographic studies of the EsA holoenzyme16 (the thermophilic equivalent of Es70), a closure of 68 was observed in the mobile domains, when EsA was complexed with fork junction DNA.17 This complex was proposed to represent the RNAP

Figure 2. Surface views of cryo-EM reconstructions of ˚ . Views (a) E7, (b) Es54 (8) and (c) Es54-DNA filtered to 21 A are facing the catalytic centre marked by an asterisk (*) showing the change in dimension of the active centre. Domain 4 of the b subunit is contained within the ellipse. bD, b downstream lobe; b 0 C, b 0 clamp domain. The arrow (/) indicates the location of the secondary channel.

s54-RNA Polymerase Holoenzyme–DNA Complex

structure in the open promoter complex.17 Figure 1(a) and (c) show the subunit organisation of Es54-DNA (RNAP closed complex) compared to the crystal structure EsA-DNA (Figure 1(b) and (d); proposed to represent the RNAP open complex). In both these cases, the DNA appears to occupy a position outside of the active centre (labelled with an asterisk (*) in Figure 2(a)). Earlier footprinting studies on complexes of promoter DNA with Es54 support this observation, since the early melted DNA shows protection from K33 to K5 only, suggesting that the C1 DNA is not at the active site.12 The extra density (marked by a black square in Figure 1(a)) found in the cryo-EM reconstruction of Es54-DNA, when compared to Es54, does not exist in the higher resolution structure of Es54 (see Figure 4 of Wigneshweraraj et al.9) and thus cannot be due to a change in the position of s54. At the same time, the extra density is insufficient to account for all the density expected for the 28 base-pairs of DNA protected by Es54 in DNA footprinting studies.4,18 This implies that some of the expected DNA electron density appears to be missing, possibly due to the DNA occupying a range of positions relative to Es54 rather than just one rigid position.

203 which are not apparent in the crystal structures of bacterial RNA polymerases.16,17,21 Changes in the overall architecture of RNAP We sought a comparison of Es54 and Es54-DNA structures, determined using molecular images of similar quality and generated using the same starting model (a cryo-EM model of Es54).8 We also used a DNA probe that had been biotinylated at the K35 end, and bound to streptavidin, in an

Conformational changes in the mobile domains of RNA polymerase The binding of the core enzyme (Figure 2(a)) to s54 results in a change in the position of domain 4 of the b subunit (residues 575–660; marked with an ellipse in Figure 2(b)).8 In the presence of heteroduplex DNA (Figure 2(c)), this domain 4 region appears to open up, possibly to accommodate any emerging DNA:RNA duplex. The conformational state of domain 4 of the b subunit within the promoter complex (Figure 2(c)) resembles that observed in structures of the native E. coli core enzyme complexed to the transcription elongation factor GreB derived from cryo-EM of helical crystals.19 Docking of the EsA X-ray structure into the Es54-DNA cryo-EM density using the program Situs,20 revealed that this region cannot be the equivalent of the “bridge helix” (data not shown).21 The b 0 clamp domain and b lobe are known to be involved in the later stages of open complex formation,22 and the residues constituting the b jaw, near the b 0 clamp domain are known to behave differently in Es54 and in the Es70 complex. However, the appearance of these minor conformational changes may be influenced by differences in quality of the initial electron images of the Es54 and the Es54-DNA complexes. No density exists in the crystal structure of EsA DNA at the position equivalent to that of domain 4 of the b subunit in our reconstruction. The cryo-EM structure of the Es54-DNA complex represents a more complete structure of the enzyme, as it does contain the carboxy-terminal domains of the a subunits,10

Figure 3. (a) Surface view of the crystal structure of T. aquaticus EsA-DNA (PDB code 1L9Z); the black line indicating the corresponding location of the 3D sections of the EM reconstructions (b). Holoenzyme subunits are colored according to the scheme used in Figure 1. (b) Section through the filtered 3D density of Es54 (left) and Es54 DNA-streptavidin (right). (The b subunit is indicated by a white, unfilled arrow; b 0 , filled arrow. The central cavity within sections contains the active centre of RNAP.) The reconstituted Es54 and Es54-DNA tagged with streptavidin were studied using frozen hydrated specimens stained with 4% (w/v) uranyl acetate. Transmission electron microscopy data were collected with a Philips CM300 FEG microscope at liquid nitrogen temperature. Micrographs were collected using a range of defocus values (1.9 mm–3 mm) underfocus. Eleven micrographs for Es54 and 38 micrographs for Es54-DNA-streptavidin were selected for subsequent image processing. These were digitised using a Nikon 8000 scanner with a step size of ˚ on the specimen scale). A total of 4800 6.35 mm (2.057 A particles manually for Es54, and 3300 for Es54-DNAstreptavidin, were selected of which over 1500 from each were used for the final reconstructions. Subsequent image processing was applied as described in the legend to Figure 2. For the final representation of the results, a bandpass filter was applied to the 3D structures to suppress ˚ and high-frelow-frequency components below 160 A ˚. quency components above 25 A

204 attempt to localise the DNA. Electromobility shift assays revealed that the addition of streptavidin to the DNA did not affect the formation of the Es54-DNA complex (data not shown). The latter complex was studied using images obtained by cryo-negative staining.23,24 Figure 3 shows one section through the 3D reconstructions displaying the overall changes in the architecture of the Es54 enzyme upon DNA binding. The changes observed in the 40 central sections (out of a total of 100 sections) through the 3D reconstruction can best be viewed in the movie supplied as supplementary material (see Supplementary Data). Overall, RNAP bound to DNA adopts a more closed conformation compared to the holoenzyme. Specifically, the b and b 0 mobile subunits move towards the active centre (Figure 3(b)). Certain parts of the domains change dimensions; other regions remain blurred, indicating flexibility of the domains. This was observed also in the individual class averages (data not shown). In particular, the b 0 clamp domain and b lobe demonstrate significant conformational changes (Figure 3(b) and Supplementary Data). In the EsA DNA holoenzyme/fork junction crystal structure,17 the RNAP clamp domain rotates in, towards the RNAP channel by some 38. Our structures derived from cryo-negative staining, suggest that the mobile domains also move towards the RNAP channel in the closed complex. The Es54-DNA structure described here provides an impression of the bacterial RNAP in a closed promoter complex configuration. Domains of the b and b 0 subunits of RNAP from E. coli change positions, probably to facilitate the interaction of promoter DNA with the s54 factor, as shown in footprinting studies.13 This may help place the DNA template at the active site.25 The changes observed here are not due to the positioning of DNA within the cleft of the enzyme (which would be expected in the intermediate or open complex,9 but not in the closed complex). DNA in the closed complex is associated with the s54 factor, not the core enzyme.26 Moreover, recent photochemical cross-linking studies show that the DNA sequence from K5 to C1 does not cross-link to E or s54 in Es54 closed complexes (P.C. Burrows et al., unpublished results). Furthermore, DNaseI footprints of unactivated closed complexes are short and do not extend beyond K5.13,26 Changes in the locations of the b 0 clamp domain and b lobe27,28 upon addition of DNA, indicate that they have a role in establishing different conformational states of RNAP upon association with promoter DNA; they are known to be involved in the later stages of open complex formation, and the changes we see may represent a preparation for such later roles.17 The transition of the transcription machinery from a closed state to an open state is facilitated by ATP hydrolysis. It remains to be seen what changes occur in core RNAP for it to accommodate the s 54 -enhancer binding activator–protein complex.29

s54-RNA Polymerase Holoenzyme–DNA Complex

Acknowledgements We are most grateful to B. Gowen (currently at the Department of Biology, University of Victoria, Canada) for collecting the initial cryo-EM data sets. We thank T. Pape, P. da Fonseca, S.R. Wigneshweraraj, and P.C. Burrows for comments on the manuscript, and J. Schumacher for advice on DNA purification. This study was supported by BBSRC project grants to M.B. (B13442) and M.v.H. (B13016). R.J.H. was funded by a BBSRC PhD studentship (no 6235). The work was conducted in the Centre for Biomolecular Electron Microscopy/ Centre for Structural Biology, Imperial College London.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2005.09.057 The supplementary material consists of a movie, best viewed using Quicktime player in the loop option. The movie displays the 40 central sections (out of a total of 100) of the 3D reconstructions derived from cryo-EM of stained samples of Es54 (open structure) and Es 54 -DNA-streptavidin (closed structure). The sections are displayed prior to filtering and masking.

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Edited by W. Baumeister (Received 28 July 2005; received in revised form 19 September 2005; accepted 20 September 2005) Available online 5 October 2005