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RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex

2006; Springer Nature; Volume: 25; Issue: 16 Linguagem: Inglês

10.1038/sj.emboj.7601261

1460-2075

Sebastian P. Maurer, Jürgen Fritz, Georgi Muskhelishvili, Andrew Travers,

Genomics and Chromatin Dynamics

Article3 August 2006free access RNA polymerase and an activator form discrete subcomplexes in a transcription initiation complex Sebastian Maurer Sebastian Maurer International University Bremen, Bremen, Germany Search for more papers by this author Jürgen Fritz Jürgen Fritz International University Bremen, Bremen, Germany Search for more papers by this author Georgi Muskhelishvili Georgi Muskhelishvili International University Bremen, Bremen, Germany Search for more papers by this author Andrew Travers Corresponding Author Andrew Travers MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Sebastian Maurer Sebastian Maurer International University Bremen, Bremen, Germany Search for more papers by this author Jürgen Fritz Jürgen Fritz International University Bremen, Bremen, Germany Search for more papers by this author Georgi Muskhelishvili Georgi Muskhelishvili International University Bremen, Bremen, Germany Search for more papers by this author Andrew Travers Corresponding Author Andrew Travers MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Sebastian Maurer1, Jürgen Fritz1, Georgi Muskhelishvili1 and Andrew Travers 2 1International University Bremen, Bremen, Germany 2MRC Laboratory of Molecular Biology, Cambridge, UK *Corresponding author. MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. Tel.: +44 1223 402419; Fax: +44 1223 412142; E-mail: [email protected] The EMBO Journal (2006)25:3784-3790https://doi.org/10.1038/sj.emboj.7601261 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using high-resolution atomic force microscopy (AFM) we show that in a ternary complex of an activator protein, FIS, and RNA polymerase containing the σ70 specificity factor at the Escherichia coli tyrT promoter the polymerase and the activator form discrete, but connected, subcomplexes in close proximity. This is the first time that a ternary complex between an activator, a σ70 polymerase holoenzyme and promoter DNA has been visualised. Individually FIS and RNA polymerase wrap ∼80 and 150 bp of promoter DNA, respectively. We suggest that the architecture of the ternary complex provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator. Introduction DNA–protein interactions are central to the regulation of the transcription of genetic information from DNA to RNA. The process of transcription initiation requires the binding of RNA polymerase to well-defined DNA promoter regions. This binding is in many cases facilitated by activator proteins that bind within the promoter regulatory region (Browning and Busby, 2004). Yet the general spatial organisation of single ternary complexes of RNA polymerase and activator proteins at a DNA promoter region is unknown. The Escherichia coli tyrT promoter is typical of the highly active stable RNA promoters in this organism and contains three binding sites for the activating DNA-binding protein FIS upstream of the core promoter (Muskhelishvili et al, 1997). The FIS sites are in helical phase and have been proposed to facilitate the wrapping of upstream DNA around RNA polymerase (Muskhelishvili et al, 1997). In this paper we have visualised the wrapping of promoter DNA by FIS and RNA polymerase both individually and together in the ternary complex. FIS wraps the tyrT upstream activating sequence (UAS) We used atomic force microscopy (AFM) to visualise single protein–DNA complexes immobilised on mica. Imaging was performed in air in tapping mode (see Materials and methods). For each type of complex several hundred images were recorded with different AFM cantilevers to obtain statistically valid information on the shape and structure of the different complexes. The association of three FIS dimers with the three FIS binding sites in the tyrT UAS upstream of the core promoter (Lamond and Travers, 1983) is a highly cooperative event with a Hill coefficient of ∼3 on linear DNA (Pemberton et al, 2002), a value that is indicative of the formation of a highly organised complex. On visualisation we observed that FIS formed a compact complex at the UAS and that its binding reduced the contour length of the DNA by 29 nm, corresponding to ∼87 bp, a value very similar to the length of the UAS (Figures 1A and D, 2 and 3A and B) and to the theoretically predicted value of 81 bp (Herman, 1996). We conclude that the FIS complex wraps DNA. To estimate more precisely the position of the FIS binding site, we determined the average lengths of the unbound DNA to the left and right arms of the complex (Figure 2A and B). These measurements in principle define two possible positions, one including the whole UAS region and a second downstream of the core promoter. However, since one boundary of the former position coincides with one of the ternary complex boundaries (see below), we conclude that FIS forms a compact complex at the UAS. Compared with RNA polymerase the approximate size of this complex is consistent with a content of at least three FIS dimers. Figure 1.Histograms of the DNA contour length distributions in nucleoprotein complexes. (A) DNA contour length distributions of the FIS–tyrT promoter DNA complexes using the 1167 bp tyrT1 template. (B) DNA contour length distributions of the RNAP–λPR DNA complexes using the 735 bp λPR promoter fragment. (C) DNA contour length distributions of the RNAP–tyrT promoter DNA complexes using the 819 bp tyrT2 template. The number of analysed complexes is indicated on the ordinate. The black bars—free DNA, grey bars—bound DNA. (D) Summary of the DNA contour length distributions and DNA shortening data. RNAP-d61 indicates the contour length distributions for complexes of RNAP with the 750 bp tyrT construct comprising the core promoter but with internally deleted UAS region. The histograms were generated by Originlab Pro 7.5 software. The curves in (A)–(C) represent the Gaussian fitting over the distribution of classes. The t-test assuming a Gaussian distribution confirmed that in all cases the shift in the contour lengths is highly significant. Download figure Download PowerPoint Figure 2.Determination of the binding positions for FIS, RNAP and the ternary complex on the tyrT promoter DNA. (A) Spatial localisation map. The left (promoter upstream) and right (promoter downstream) arms are indicated for each the FIS and the RNAP complexes. For the ternary FIS–RNAP–promoter DNA complex, for determination of binding position only the long left arm was used. The spatial organization of the initiation start point, the −10 and −35 hexamers and the three FIS binding sites is indicated. Promoter fragments of different lengths were used for right arm measurements for FIS (tyrT1) and RNAP (tyrT2). (B) Summary of the binding position determination data. Download figure Download PowerPoint Figure 3.AFM images of the nucleoprotein structures formed by FIS, RNAP and the ternary polymerase–FIS–promoter complexes. The respective 2 × 2 μm overviews (A, C, E—the scale bar is 200 nm) and 240 × 240 nm magnified images (B, D, F—the scale bar is 100 nm) in an angled view (left panel) and top view (grayscale, right panel) are shown. Different tips were used. Download figure Download PowerPoint RNA polymerase wraps ∼150 bp of tyrT promoter DNA Previous studies have demonstrated that RNA polymerase containing the σ70 specificity factor interacts directly with the UAS of the tyrT promoter, both protecting at least 130 bp upstream of the transcription start site from DNase I cleavage (Travers et al, 1983) and photosensitising specific bases between positions −120 and −130 (Pemberton et al, 2002). Although the untwisting of DNA at the startpoint of the tyrT promoter and consequent formation of salt-stable complexes requires the initiating triphosphates the binding of the upstream region is independent of the presence of these nucleotides (Debenham, 1979; Travers et al, 1983; Auner et al, 2003). To visualise the enzyme bound at the tyrT promoter by AFM, we bound polymerase to the tyrT2 DNA fragment (Figure 1C) in the absence of nucleoside triphosphates at low temperatures. The appearance of these complexes was variable (Figures 3B, D, F and 4A). The complexes formed under these conditions would represent closed, or possibly nucleated, but not open, transcription initiation complexes. The polymerase–promoter complex reduced the contour length of DNA by 52 nm corresponding to ∼150–160 bp (Figure 1D), a value that is similar to the complete tyrT promoter including the UAS region and is in good agreement with previous footprinting studies (Travers et al, 1983; Pemberton et al, 2002) but is considerably larger than values observed at other promoters (Rivetti et al, 1999; Shin et al, 2005). Measurements of the unbound DNA arms of the polymerase–promoter complex indicated that RNA polymerase binding site included the core promoter and the upstream region in the vicinity of FIS binding site III (Figure 2A and B). Under our conditions, we could accurately reproduce the reduction in contour length consequent on binding polymerase to the λPR as recently determined (Rivetti et al, 1999; Figure 1B). In addition, we measured the contour lengths of complexes formed by RNAP with a tyrT construct in which the sequence upstream of position −61, that is including the UAS, is deleted. We observed that for this construct the reduction in contour length was only 22 nm corresponding to ∼66 bp (see RNAP-d61 in Figure 1D). This value corresponds well to the length of the core promoter including the UP element between −48 and −56. We conclude that the complete tyrT promoter binds a single molecule of polymerase holoenzyme and at this promoter the DNA is wrapped around the enzyme as originally hypothesised by Buc (1986). The data indicate the extensive DNA wrapping by RNA polymerase requires the presence of the UAS. Figure 4.Heterogeneity of binary polymerase–promoter and ternary polymerase–FIS–promoter complexes. (A) Binary RNAP–promoter complexes with protruding DNA loops (cf Figures 3B, D and F). (B–D) Three different types of ternary complexes. In (B, C), the promoter-downstream DNA is wrapped to different extents but the promoter-upstream DNA lengths are similar and the FIS and RNAP subcomplexes are closely associated. In (D), the path of DNA connecting the two subcomplexes is seen. Note the left-handed toroidal supercoil stabilised by smaller subcomplex (D, right panel). All magnified images (240 × 240 nm, the scale bar is 100 nm) are shown in both an angled view (top panels) and top view (grayscale, bottom panels). (E) Section measurements of the height of the connections between the FIS and RNAP subcomplexes shown in (B). The white line represents the path of the section. (F) The red arrows indicate the peaks corresponding to free DNA and the connections between the subcomplexes. In both cases the connections between the subcomplexes have greater height and both single and double peaks indicative of single (upper panel) or double (lower panel) connection can be seen (240 × 240 nm2 magnification). (G) The model of the binary RNAP–promoter complex with protruding DNA loop shown in (A). (H) A model of the ternary FIS–polymerase–promoter DNA complex. (1) Binding of several FIS dimers (pink and yellow) bends the DNA (grey rod) in the subcomplex; (2) rotation of the FIS subcomplex relative to RNA polymerase (grey) brings the upstream DNA in close vicinity of polymerase; (3) binding of polymerase to upstream DNA and formation of FIS polymerase contacts stabilises a DNA microloop. Formation of this latter in absence of FIS can be facilitated by negative supercoiling of DNA, leading to structures exemplified in (G). Download figure Download PowerPoint Structure of ternary polymerase–FIS–promoter complex Not only is the binding of FIS to the tyrT UAS highly cooperative but also the dependence of transcription on FIS concentration in vitro is sigmoidal, again with a Hill coefficient of ∼3 (Pemberton et al, 2002). This correspondence implies that the structure of the FIS subcomplex is either maintained in the polymerase–FIS–promoter ternary complex or is at least an obligate intermediate in the formation of this initiation complex. Visualisation of the ternary complex revealed that the FIS subcomplex remained as a discrete assembly but in close proximity to an RNA polymerase molecule (Figures 3E and 4B). The protein components of these ternary complexes were identified by height, that of the FIS subcomplex being normally approximately half that of the polymerase. The location of this ternary assembly corresponds well to the locations of the complexes of FIS and polymerase by themselves (Figure 2A). In several examples of this complex, we see distinct single or double connections between the polymerase and the FIS assembly (see e.g. Figures 4E and F), but we have no indication of their nature except that in all ternary complexes their height is greater than that of a single DNA duplex. The morphology of the ternary complexes is more variable than that of either the polymerase–DNA or the FIS–DNA complexes alone. In many images of the ternary complex, the entering and exiting arms of DNA emerge from the combined polymerase–FIS complex suggesting that in these complexes the promoter DNA is wrapped around the whole complex. However, in others one of the contacts, probably the upstream one, between the polymerase and the DNA is no longer apparent and the DNA separates from the complex in the vicinity of FIS. In other complexes we observe completely separate complexes of FIS and polymerase (Figure 4D), while in yet others the height of the FIS peak differs from that in other ternary complexes (Figure 4C). While this variability in detail could be in part due to the variations in the AFM tip shape, it would also be consistent with previous results indicating that the ternary complex, even in the absence of the nucleoside triphosphates, is highly dynamic (Muskhelishvili et al, 1997; Pemberton et al, 2002) and that FIS can accrete to an already established complex of three FIS dimers with the UAS (Muskhelishvili et al, 1995). Nevertheless, the primary organisation into two discrete complexes is invariant and we infer that this represents the structural basis for transcriptional activation at the tyrT promoter. The formation of the ternary complexes was dependent on the prior or simultaneous addition of polymerase relative to FIS and was not observed when FIS was added before polymerase (data not shown). Implications We have shown that FIS and RNA polymerase form discrete, but connected, complexes at the tyrT promoter. To our knowledge this is the first time that a complex between an activator and a σ70 polymerase holoenzyme has been visualised. Nevertheless, the discrete character of the activator and polymerase complexes is similar in principle to that observed at the glnA promoter between the NtrC activator and a σ54 polymerase holoenzyme (Rippe et al, 1997). We conclude that by positioning the activator in close proximity to the polymerase, this architecture provides a general paradigm for the facilitation of direct, but weak, interactions between polymerase and an activator. The cooperative formation of the FIS–UAS complex and its apparent conservation in the ternary complex argue strongly that the assembly of three FIS dimers constitutes the functional activating unit at the tyrT promoter. This conclusion is supported by previous demonstrations that a single proximal FIS site is insufficient for optimal activation of this promoter (Muskhelishvili et al, 1997). RNA polymerase cooperatively recruits FIS to this proximal site (Muskhelishvili et al, 1995), suggesting that the polymerase itself can facilitate, but is not essential for, the formation of the FIS subcomplex containing three dimers. In vivo it is likely that other abundant nucleoid-associated proteins bind to the UAS so that the occupancy of the UAS by FIS is determined by dynamic competition between activating (polymerase and FIS) and repressing proteins. In vivo and in vitro the tyrT promoter as well as the similar rrnA P1 promoter are strongly dependent on high negative superhelicity of DNA for optimal transcription (Lamond, 1985; Free and Dorman, 1994; Rochman et al, 2002). In vivo in the presence of FIS the activity of these promoters is buffered against variations in superhelical density, a phenomenon that requires the binding of FIS to multiple sites in the UAS (Rochman et al, 2002; Auner et al, 2003). We suggest that FIS stabilises the left-handed wrapping of DNA in the ternary complex and activates initiation by a slight rotation of the FIS complex relative to polymerase thereby applying torque to the enzyme (Figure 4H). This would result in the untwisting of the −10 region as envisaged in our model for torsional transmission (Muskhelishvili and Travers, 1997; Muskhelishvili et al, 1997). The promoters of several other stable RNA genes, including most of those encoding rRNA (Hirvonen et al, 2001) contain multiple FIS sites in the UAS region. These are often, as in tyrU and rrnB P1, in helical register and would be expected to form compact structures related to those we observe at the tyrT promoter. Nevertheless some of these promoters, for example leuV, contain only a single FIS binding site in the UAS (Ross et al, 1999). In such cases we would expect that a compact assembly cannot be formed by FIS alone. In contrast at the rrnA P1 promoter, FIS binds to a strong far upstream site at −225 and in so doing constrains an additional negative supercoil (Rochman et al, 2002). We suggest that in this case the FIS assembly contains more FIS dimers than at the tyrT UAS. Another transcriptional activator that, like FIS, bends DNA strongly is the CRP dimer (catabolite repressor protein). At the lac promoter, CRP binds in the same helical register as FIS (Gaston et al, 1990) and also, like FIS, stabilises a DNA bend between the polymerase contacts at the −35 hexamer and in the upstream region (Buckle et al, 1992). CRP has been inferred to stabilise a similar structure at the malT promoter in vivo (Eichenberger et al, 1997). Contacts between FIS and RNA polymerase as well as those between CRP and polymerase are known to be mediated, at least in part, by the CTDs of the α-polymerase subunits (Murakami et al, 1997; Meng et al, 2001; Benoff et al, 2002). We suggest that in the absence of an activator an α-CTD contacts FIS site III in an analogous manner to its upstream contacts at the lacUV5 promoter (Davis et al, 2005; Ross and Gourse, 2005). In accord with this view, it has recently been shown by AFM that the extensive wrapping of RNA polymerase with the λPR promoter requires the interaction of an upstream sequence with the α-CTD (S Cellai, N Vannini, N Naryshkin, RH Ebright. and C Rivetti, personal communication). In contrast in the presence of an activator the α-CTDs bridge between polymerase and activator bound as distinct protein–DNA complexes. Materials and methods Proteins FIS was purified according to the protocol of Koch and Kahmann (1986). RNA polymerase was obtained from Epicentre. DNA fragments The template DNA fragments were generated from plasmids ptyrTlac and pTyrTd61 (Auner et al, 2003) by PCR using Pfu polymerase (Promega). PCR products were purified with Qiagen Qiaquick® Gel Extraction kit. The DNA was eluted with a 20 mM HEPES buffer, pH 8.0. Protein–DNA binding RNAP–DNA complexes were formed by mixing equimolar amounts of protein and DNA (1.7 μM final concentration) in 20 μl AFM-buffer (20 mM HEPES pH 8.0, 50 mM KCl, 0.005% Tween, 2 mM NiCl2) at room temperature. In all reactions containing FIS, the used molar ratio of FIS to DNA was 2.65:1. To facilitate ternary complex formation (Muskhelishvili et al, 1995), the RNA polymerase was added first to the DNA (RNAP to DNA molar ratio of 0.8:1) and FIS was added afterwards (FIS to DNA molar ratio of 2.65:1). The reaction mixture was incubated for 5 min at 4°C and subsequently transferred to a freshly cleaved mica disc (Plano Gmbh, Wetzlar). After 10 min at 4°C, the mica disc was rinsed three times with 1 ml distilled water and dried for 20 s under a weak flux of nitrogen. AFM imaging Images were acquired with a Multimode atomic force microscope equipped with a Nanoscope IIIa controller (Veeco Instruments GmbH, Germany), operating in Tapping Mode in air using a J-scanner and RTESP silicon cantilevers. Images of 512 × 512 pixels with a scan size of 2 × 2 μm were acquired at scan frequencies between 2 and 3 Hz. AFM images were processed by using the NanoScope Image software (version 5.12r5; Veeco Instruments Inc., Santa Barbara, CA, USA). Contour lengths of DNA molecules were determined manually using Image J software (version 1.32j by Wayne Rasband, NIH, USA). Statistical analysis A Gaussian fitting over the distribution of classes and Student's t-tests to verify the significance of DNA contour length change were calculated for all data sets. The confidence limits are presented in Figure 1D. Acknowledgements This work was supported by the Grant DFG-MU-2FIS from Deutsche Forschungsgemeinschaft to GM. 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Matthew Scott, who took this picture in October 2005, is a Principal Investigator of the Department of Developmental Biology at Stanford University. Visit his lab at http://scottlab.stanford.edu Volume 25Issue 1623 August 2006In this issue FiguresReferencesRelatedDetailsLoading ...