Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA
2006; Springer Nature; Volume: 25; Issue: 7 Linguagem: Inglês
10.1038/sj.emboj.7601050
ISSN1460-2075
AutoresRebekka O. Sprouse, Michael Brenowitz, David Auble,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle16 March 2006free access Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA Rebekka O Sprouse Rebekka O Sprouse Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Michael Brenowitz Michael Brenowitz Department of Biochemistry, The Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author David T Auble Corresponding Author David T Auble Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Rebekka O Sprouse Rebekka O Sprouse Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Michael Brenowitz Michael Brenowitz Department of Biochemistry, The Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author David T Auble Corresponding Author David T Auble Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA Search for more papers by this author Author Information Rebekka O Sprouse1, Michael Brenowitz2 and David T Auble 1 1Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA 2Department of Biochemistry, The Albert Einstein College of Medicine, Bronx, NY, USA *Corresponding author. Department of Biochemistry and Molecular Genetics, University of Virginia Health System, 1300 Jefferson Park Avenue, Room 6213, Charlottesville, VA 22908-0733, USA. Tel.: +1 434 243 2629; Fax: +1 434 924 5069; E-mail: [email protected] The EMBO Journal (2006)25:1492-1504https://doi.org/10.1038/sj.emboj.7601050 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mot1 is a conserved Snf2/Swi2-related transcriptional regulator that uses ATP hydrolysis to displace TATA-binding protein (TBP) from DNA. Several models of the enzymatic mechanism have been proposed, including Mot1-catalyzed distortion of TBP structure, competition between Mot1 and DNA for the TBP DNA-binding surface, and ATP-driven translocation of Mot1 along DNA. Here, DNase I footprinting studies provide strong support for a ‘DNA-based’ mechanism of Mot1, which we propose involves ATP-driven DNA translocation. Mot1 forms an asymmetric complex with the TBP core domain (TBPc)–DNA complex, contacting DNA both upstream and within the major groove of the TATA Box. Contact with upstream DNA is required for Mot1-mediated displacement of TBPc from DNA. Using the SsoRad54–DNA complex as a model, DNA-binding residues in Mot1 were identified that are critical for Mot1–TBPc–DNA complex formation and catalytic activity, thus placing Mot1 mechanistically within the helicase superfamily. We also report a novel ATP-independent TBPc displacement activity for Mot1 and describe conformational heterogeneity in the Mot1 ATPase, which is likely a general feature of other enzymes in this class. Introduction Mot1 is an essential transcriptional regulator in Saccharomyces cerevisiae that directly interacts with the TATA-binding protein (TBP) in solution and when both proteins are bound to promoter DNA (Davis et al, 1992; Auble et al, 1994; Poon et al, 1994; Adamkewicz et al, 2001; Darst et al, 2001; Pereira et al, 2003). A remarkable property of Mot1 is its ability to utilize the energy derived from the hydrolysis of ATP to displace TBP bound to a TATA Box sequence on DNA. This activity rationalizes Mot1's function as a transcriptional repressor (Auble et al, 1994). The requirement for Mot1 to activate transcription of some genes (Collart, 1996; Madison and Winston, 1996; Prelich, 1997; Andrau et al, 2002; Dasgupta et al, 2002) can also be explained at least in part by the ability of Mot1 to eject inactive TBP-containing complexes from activated promoters (Geisberg et al, 2002; Dasgupta et al, 2005). Mot1 is a member of the Snf2/Swi2 ATPase family (Eisen et al, 1995). Enzymes in this class participate in all of the fundamental processes of DNA metabolism, utilizing ATP hydrolysis to alter contacts between proteins and DNA (Vignali et al, 2000). Many enzymes in this family reposition nucleosomes, structurally alter them, or remove them entirely from DNA (Becker and Horz, 2002; Narlikar et al, 2002). The changes that occur in nucleosomal DNA follow multiple cycles of ATP hydrolysis driving changes in DNA twist or bulge propagation over the surface of the nucleosome (Flaus and Owen-Hughes, 2003). These observations have given rise to the idea that these ATPases function as molecular motors. While the utilization of ATP hydrolysis to drive changes in DNA topology is well established, relatively little is known about how the chemical energy from ATP hydrolysis is used to generate mechanical force driving disruption of protein–DNA interactions (Smith and Peterson, 2005). Mot1-catalyzed dissociation of the TBP–DNA complex affords a simplified system for defining how this chemical–mechanical coupling occurs. Mot1 neither translocates processively along DNA nor functions by propagating changes in DNA structure (Auble and Hahn, 1993; Auble and Steggerda, 1999; Adamkewicz et al, 2001; Darst et al, 2001). Mot1 stabilizes TBP binding to nonconsensus TATA sequences in the absence of ATP (Gumbs et al, 2003; Klejman et al, 2005). Thus, rather than manipulating DNA structure, Mot1 may use ATP hydrolysis to distort TBP structure (Adamkewicz et al, 2001; Darst et al, 2003; Gumbs et al, 2003). Such a mechanism would represent a novel use of such an ATPase. Yeast Mot1 and its human homolog, BTAF1, compete with DNA for binding to TBP (Pereira et al, 2001; Darst et al, 2003). This competition has given rise to an alternative, but not mutually exclusive, suggestion that the interaction between Mot1 and the TBP DNA-binding surface contributes to the Mot1/BTAF1 regulatory mechanism (Pereira et al, 2001; Darst et al, 2003). A third idea posits that Mot1, as a member of the helicase superfamily (Eisen et al, 1995), uses a mechanism of ATP-driven DNA translocation to disrupt the interaction between TBP and DNA (Darst et al, 2001). In support of the DNA translocation model, the recent crystal structures of the Snf2/Swi2-related ATPase Rad54 from zebrafish (Thoma et al, 2005) and Sulfolobus (Durr et al, 2005) revealed structural similarity between the ATPase and helicase catalytic cores (Durr et al, 2005). However, in the SsoRad54–DNA complex, the ATPase domains were in an unexpected arrangement in which residues required for ATP binding and hydrolysis were misaligned and the ATP binding cleft was unformed (Durr et al, 2005). Durr et al (2005) proposed a model reconciling this structure with helicase activity in which domain rotation induced by ATP binding and hydrolysis results in translocation of the enzyme along DNA. In this study, we test a model for Mot1 action based on the SsoRad54–DNA structure and conclude that Mot1 is a nonprocessive DNA-translocating enzyme. These results also provide support for the biochemical significance of ATPase domain rotation observed in the SsoRad54–DNA crystal structure. Moreover, a novel ATP-independent displacement activity of Mot1 was observed, which appears to arise at least in part from conformational variability in the ATPase and is likely a general feature of enzymes in this class. Results These studies employed purified, bacterially expressed, yeast TBP and highly purified Mot1, which was obtained from a yeast overexpression strain (Figure 1A; Darst et al, 2001). Using these proteins, TBP–DNA and Mot1–TBP–DNA ternary complexes were detectable by electrophoretic mobility shift assays (Figure 1B and C). The addition of unlabeled competitor DNA demonstrated that preformed TBP–DNA complexes dissociated over the time course, whereas the detectable ternary complexes were markedly more stable. In contrast, addition of ATP resulted in Mot1-catalyzed displacement of both proteins from DNA in less than 2 min. While these assays confirm the general features of Mot1's activity, quantitative studies are problematic using this method because of the time required to load the gels and the instability of TBP–DNA complexes during electrophoresis (Hoopes et al, 1992). We therefore turned to DNase I footprinting, a technique that circumvents these limitations and identifies the regions of protein contact on the DNA in solution (Galas and Schmitz, 1978). Figure 1.(A) A representative 12% protein gel showing the purified wild-type Mot1 protein used in these studies. The gel was stained with Coomassie blue and molecular weight standards are shown on the left. (B) Electrophoretic mobility shift assay using radiolabeled DNA (<1 nM) combined with 10 nM TBP where indicated. Unlabeled competitor TATA DNA was added prior to the addition of TBP (lane 3; 1st), or was incubated with preformed TBP–DNA complexes for the indicated times prior to loading on the gel (lanes 4–7). (−) indicates that the component was not added. The positions of the free DNA and TBP–DNA complex are shown on the left. Quantitation of the TBP–DNA complex revealed a dissociation half-time of ∼17 min (not shown). (C) An electrophoretic mobility shift experiment in which TBP (10 nM) was preincubated with radiolabeled DNA (<1 nM) as in (A), followed by addition of Mot1 (10 nM) to all reactions for 2 min. In lanes 3, 4, 7, and 8, unlabeled competitor TATA DNA was then added with or without 25 μM ATP for the indicated times prior to loading on the gel. Competitor DNA was added prior to TBP in the reactions in lanes 2 and 6. The positions of the free DNA, TBP–DNA, and Mot1–TBP–DNA complexes are indicated. Download figure Download PowerPoint Structural organization of the Mot1–TBPc–DNA complex DNase I footprint titration experiments were conducted in which increasing concentrations of TBP core domain (TBPc) were titrated against radiolabeled DNA bearing the high-affinity TATA Box sequence TATAAAAG (Figure 2A and B). TBPc provides full function in yeast cells (Reddy and Hahn, 1991) and is displaced by Mot1 from DNA indistinguishably from full-length TBP (Auble and Hahn, 1993; Darst et al, 2001). The tighter DNA binding affinity of TBPc compared to full-length TBP improved the reproducibility and robustness of DNase I protection and thereby facilitated this quantitative analysis. The measured Kd of 3.8±0.6 nM measured for the TBPc–DNA interaction and the crispness of the observed protection pattern demonstrate high affinity and specificity of this interaction under these experimental conditions. Figure 2.(A) An autoradiogram showing a DNase I footprint experiment in which TBPc was titrated to DNA. Lane 1 shows free DNA. Reactions in lanes 2–9 contained 0.25, 0.75, 2.0, 3.0, 5.0, 7.5, 12.6, or 25.1 nM TBPc, respectively. (B) Analysis of the experiment in (A), yielding an apparent Kd of 3.8±0.6 nM for the TBPc–DNA interaction. Quantitation was performed by block analysis (Brenowitz et al, 1986) of the TATA band intensities normalized for differences in lane loading using the bands in the indicated region of the gel. (C) An autoradiogram showing a DNase I footprint titration experiment in which Mot1 was titrated to the TBPc–TATA complex. Lanes 1 and 8 show the free DNA. Lanes 2–7 included TBPc at 8.8 nM and Mot1 at 0.0, 0.4, 1.2, 4.1, 6.1, or 8.1 nM Mot1 for lanes 2–7, respectively. The positions of those upstream bands whose DNase I reactivity reflects only Mot1 binding are shown to the left of the autoradiogram (see Figure 3). A decrease in TATA Box protection upon addition of Mot1 was also observed; this is quantitated in Figure 3. (D) An isotherm calculated from (C) for the binding of Mot1 to the TBPc–DNA complex. The changes in density of the bands corresponding to nucleotides 37, 38, 42, and 43 quantitated individually were globally analyzed against the Langmuir polynomial to yield a best-fit value of Kd=1.5±0.5 nM as depicted in the simulated curve. Download figure Download PowerPoint Titration of Mot1 to the TBPc–DNA complex resulted in an extension of the DNase I footprint upstream from the TATA Box (Figure 2C). Quantitation of the bands uniquely protected from DNase I by Mot1 yields an apparent Kd of 1.5±0.5 nM for its association with the TBPc–DNA complex (Figure 2D). This reaction includes both intrinsic (i.e., Mot1–DNA interactions) and cooperative (i.e., Mot1–TBPc interactions) contributions since Mot1 does not detectably bind to DNA in the absence of TBPc (Ackers et al, 1983; Darst et al, 2001; and data not shown). To probe the Mot1–TBPc–DNA ternary complex in detail, autoradiograms such as that shown in Figure 2C were quantitated with single-nucleotide resolution (Takamoto et al, 2004; Das et al, 2005). The extent of DNase I protection for the DNA alone, TBPc–DNA, and TBPc–Mot1–DNA complexes is shown in Figure 3. The TBPc footprint extends from nucleotides 36 to 19, bracketing the TATA Box. The addition of Mot1 yielded unique upstream protections at nucleotides 37, 38, 42 and 43. Owing to band compression at more upstream positions, the upstream edge of the Mot1 footprint was not determined in this analysis, but the overall pattern of upstream protection closely resembles that reported previously (Darst et al, 2001). Neither protein affected the DNase I reactivity of nucleotides downstream of 19. These observations are consistent with both proteins directly contacting the DNA; this conclusion is supported by crosslinking studies that are discussed below (Figure 8). We also noted that the extent of DNase I protection within the TATA Box decreased as Mot1 binding increased (Figure 2C and data not shown). This decrease in TATA Box protection was observed for every nucleotide protected by TBPc (Figure 3). The Mot1-mediated increase in DNase I reactivity within the TATA Box could result from a conformational change in which TBPc is partially lifted from the TATA Box, or alternatively, some TBPc may be displaced by Mot1 in the absence of ATP hydrolysis. Figure 3.A histogram showing quantitation of the relative intensity of the electrophoretic bands within and surrounding the TATA Box for DNA alone (blue), TBPc alone (black), and TBPc+Mot1 (orange) quantitated with single-nucleotide resolution. The data were derived from averaging lanes 1 and 8, or 2 and 7 of Figure 2C, respectively. The error bars on the free DNA data are the standard deviation of the average of the two lanes. The alterations in DNase I digestion pattern induced by TBP and Mot1 quantified here were observed in multiple independent experiments (not shown). Download figure Download PowerPoint Unexpected heterogeneity in the Mot1–TBPc–DNA complex To distinguish between these models, kinetics experiments were performed in which displaced TBPc was trapped with unlabeled competitor DNA, allowing dissociation to proceed to completion. In these experiments, TBPc was added to the DNA and allowed to equilibrate for 20 min, and then Mot1 was added at the indicated concentrations and allowed to equilibrate for 2 min before the addition of competitor DNA. In the absence of Mot1, the typically slow rate of TBPc dissociation was observed, following an exponential decay with a half-time of 17.9±1.8 min. This reaction was minimally affected by ATP (t=12.2±1.2 min; Figure 4A). Figure 4.(A) The rate of TBPc dissociation was monitored by DNase I footprinting in the absence (open symbols) and presence (filled symbols) of 25 μM ATP. The half-time values determined for the best-fit to a single exponential decay are 17.9±1.8 and 12.2±1.2 min, respectively. (B) The rate of dissociation of the TBPc–Mot1–DNA complex in the absence (open symbols) and presence (solid symbols and insert) of 25 μM ATP. The ATP-mediated dissociation reaction is well described by a single exponential decay characterized by a half-time of 0.91±0.09 min. In contrast, the progress curve measured in the absence of ATP is biphasic and characterized by half-times of 1.8 (−0.5, +0.7) and 92.2 (−12.0, +17.0) min and amplitudes of 0.37±0.6 and 0.63±0.5, respectively. The fit of the data to a double compared with single exponential decay was significantly better as judged by the distribution of residuals (not shown) and χ2 values (2.18 × 10−3 versus 1.26 × 10−2, respectively). Download figure Download PowerPoint When the experiment was conducted with Mot1 present and ATP added together with the competitor DNA, dissociation was fast with a half-time of 0.91±0.09 min in a reaction that is also described as an exponential decay (Figure 4B; see Supplementary Figures S1 and S2 for representative autoradiograms and ATP hydrolysis rate under these conditions). Surprisingly, in the absence of ATP, dissociation of the Mot1–TBPc–DNA complex was biphasic (Figure 4B). A fast phase characterized by a half-life of 1.8 (+1.0, −0.7) min comprises about a third of the reaction amplitude. This fast phase was followed by a slow one characterized by a half-life of 92.2 (+23.6, −16.4) min. The fast phase represents a 10-fold destabilization of the TBPc–DNA complex, whereas the slow phase represents 5-fold stabilization. Since neither rate corresponds to that observed for TBPc alone, these dissociation events are unique to the ternary complex. The uniqueness of this dissociation behavior is supported by kinetic analysis of the mot1–505 mutant protein that harbors mutations in conserved residues of the ATP-binding Walker B motif (D1408A, E1409A, H1411A; Eisen et al, 1995) destroying ATPase activity. Dissociation of mot1–505–TBPc–DNA complexes in the presence of ATP was also biphasic; the dissociation progress curve for this reaction was very similar to that of wild-type ternary complexes in the absence of ATP (compare Figure 5 with Figure 4B). Clearly, the biphasic dissociation behavior is not owing to residual Mot1 catalytic activity resulting, for instance, from ATP contamination. The biphasic ternary complex dissociation data explain why nuclease sensitivity increases in the TATA Box in proportion to increasing Mot1: Mot1 can dissociate a proportion of the TBPc–DNA complexes in the absence of ATP. These results also argue against the possibility that the increased TATA Box reactivity (Figure 2C) results from a conformational change in TBP induced by Mot1. Figure 5.The rate of dissociation of TBPc–mot1–505–DNA complexes in the presence of 25 μM ATP. The progress curve is biphasic and characterized by half-times of 1.25 (−0.37, +0.48) and 120.1 (−47.9, +227.8) min and amplitudes of 0.42±0.06 and 0.58±0.05, respectively. Download figure Download PowerPoint Perturbation of the linker connecting ATPase subdomains affects ternary complex heterogeneity The biphasic kinetic behavior suggests that in the absence of ATP, wild-type Mot1 assembles into at least two types of ternary complexes with different stabilities. While this heterogeneity could arise from conformational differences in any of the ternary complex components, recent structural studies of the homologous Snf2/Swi2 family member SsoRad54 (Durr et al, 2005) point to Mot1. The ATPase subdomains of SsoRad54 are rotated in the crystal structure ∼180° away from the positions required for establishment of the ATP binding cleft (Durr et al, 2005; Figure 6A and B, ‘open’ and ‘closed’ forms). This suggests that Snf2/Swi2 ATPase domains can undergo functionally significant rotational changes. The sequences of SsoRad54 and Mot1 align over the entire length of their ATPase domains with an expectation value of 5 × 10−71. This high degree of homology strongly supports the hypothesis that these proteins have the same overall fold and that the Mot1 ATPase domain can likewise undergo a functionally significant rotational change in conformation. Figure 6.(A) Ribbon representation of Sso–Rad54 bound to DNA (Durr et al, 2005). View along the ATP binding cleft shows domain 1 (left) and domain 2 (right) connected by a flexible hinge (bottom). Homologous residues corresponding to mutations analyzed in this study are shown as colored spheres: mot1–24 (hinge region, yellow); mot1–505 (Walker B motif, cyan); mot1–508 and mot1–509 (DNA contacting residues in domain 1A; blue and magenta, respectively). (B) Model for Mot1 ATPase conformational change. Interaction of the ATPase with DNA (thick bar) is mediated primarily by residues in domain 1A. In the open conformation, domain 2A is oriented with ATP-binding residues pointing outward. An ∼180° rotation of domain 2A establishes the ATP binding cleft between domains 1A and 2A, and swings the Snf2/Swi2-specific 1B and 2B domains in position to push on DNA during the power stroke. A hinge connects the two pairs of domains and is the location of the R1507K mutation. Model is based on the SsoRad54–DNA structure (Durr et al, 2005) and the PcrA–DNA–AMPNP structure (Velankar et al, 1999). (C) DNase I footprinting experiment with reactions that contained 8.8 nM TBPc and/or 9 nM mot1–24 as indicated. Arrow indicates upstream protection induced by mot1–24. mot1–24 alone had no effect on the DNase I digestion pattern (not shown). (D) The rate of dissociation of the TBPc–mot1–24–DNA complex in the presence of 25 μM ATP. The progress curve is biphasic and characterized by half-times of 0.23 (−0.17, +0.42) and 36.2 (−2.5, +3.1) min and amplitudes of 0.09 (−0.02, +0.03) and 0.91 (−0.03, +0.01), respectively. (E) Relative TBP-stimulated ATPase activity of wild-type Mot1, mot1–505, and mot1–24. ‘None’, no Mot1 added. Average activity±standard deviation derived from two or more assays is shown. TBP (100 nM) was present in all reactions. Download figure Download PowerPoint This hypothesis was tested by analysis of the activity of mot1–24 (Madison and Winston, 1996), a conditional mutant protein harboring a single amino-acid change within the hinge connecting the subdomains proposed to undergo rotation (Figures 6A and B). Although mot1–24 footprints with TBPc–DNA indistinguishably from wild-type Mot1 in the absence of ATP (Figure 6C), the dissociation kinetics of the mot1–24–TBPc–DNA ternary complex differ significantly from wild-type ternary complex kinetics (Figure 6D). Notably, the amplitude of the fast kinetic phase is barely distinguishable and the slow phase is ∼2.5-fold less stable than ternary complexes formed with wild-type Mot1. The absence of appreciable heterogeneity in the stability of mot1–24 ternary complexes suggests that the wild-type heterogeneity in ternary complex stability is owing to structural heterogeneity in Mot1 conformation. Because mot1–24 had no detectable ATPase activity (Figure 6E), we suggest that the linker mutation impairs conversion of the catalytically inactive ‘open’ conformation to the catalytically active ‘closed’ conformation, and that both conformational forms can interact with TBPc–DNA. Catalysis of TBPc–DNA dissociation by Mot1 requires upstream DNA If an interaction of Mot1 with upstream DNA is functionally important for Mot1-mediated catalysis of TBPc displacement, then Mot1 should not remove protein bound to a DNA template lacking the upstream footprinted sequence. Two templates with truncated upstream DNA were tested (Figure 7A; templates 5–3 and 6–1); the TBPc footprint and binding affinity were indistinguishable when compared to the full-length RW2 probe (not shown). However, significant differences were observed when Mot1 was added to preformed TBPc–DNA complexes. The control template displayed the expected ATP-dependent Mot1-mediated displacement of TBPc from the DNA (Figure 7B; RW2). Mot1 removed essentially all of the bound TBPc following 1.0 min incubation with ATP, consistent with Figure 4. In contrast, Mot1 displaced relatively little of the TBPc bound to the short DNA templates after 5.0 min ATP incubation. Figure 7.Upstream DNA is critical for Mot1's ATP-dependent dissociation activity. (A) A schematic representation of three DNA molecules possessing identical TATA and downstream DNA sequences, but different lengths of DNA upstream of the TATA Box. The length of each upstream strand is indicated in nucleotides. (B, C) The TATA Box occupancy as assayed by DNase I footprinting of TBPc and TBP, respectively, on the three DNA templates. The concentrations of TBPc, TBP, Mot1, and ATP used were 8 nM, 12 nM, 8 nM, and 25 μM, respectively. ATP was added for 1 or 5 min as indicated. Representative autoradiograms for this experiment are shown in Supplementary Figure S3. (D) Rate of dissociation of the Mot1–TBPc–DNA ternary complex in the presence of ATP. The dissociation reaction is well described by a single exponential with a half-time of 9.5 (−1.1, +1.3) min. (E) Rate of dissociation of the Mot1–TBPc–DNA ternary complex in the absence of ATP. The curve is biphasic and is characterized by half-times of 3.8 (−1.5, +2.4) and 50.7 (−10.1, +12.4) min. Download figure Download PowerPoint Kinetic experiments (Figure 7D) demonstrated that the half-time of ATP-dependent Mot1-mediated TBPc displacement from the 5–3 template was 9.5 (−1.1, +1.3) min, a value comparable to that measured in the absence of Mot1. This result confirms that the DNA upstream of the TATA Box is critical for ATP-dependent Mot1-mediated catalysis of TBPc–DNA dissociation under these experimental conditions. As expected, the requirement for upstream DNA for ATP-dependent Mot1-mediated displacement was also observed for the full-length TBP (Figure 7C). The ATP-independent displacement of TBPc from the 5–3 template by Mot1 was characterized by the biphasic progress curve observed for the long DNA (Figure 7E). Approximately 30% of the dissociation reaction occurred with a half-time of 3.8 (−1.5, +2.4) min while the remainder occurred with a half-time of 50.7 (−10.1, +12.4) min. Thus, the ATP-independent dissociation of TBPc–DNA by Mot1 does not have an upstream DNA requirement. Site-specific photocrosslinking of Mot1 to DNA To test directly the hypothesis that Mot1 intimately contacts DNA, photocrosslinking was performed to determine if Mot1 could be covalently bound to DNA probes with 5-iodouracil (5-IdU; Malkov and Camerini-Otero, 1995) clusters positioned on either strand, within or upstream of the TATA Box (Figure 8A). Irradiation of ternary complexes formed with native unsubstituted DNA at the 5-IdU-selective wavelength of 312 nm did not yield crosslinked Mot1, but crosslinking to Mot1 was detected with irradiation with 254 nm UV light, presumably via thymine residues in the probe (Figure 8B, lanes 1–3). Since 5-IdU crosslinks across the major groove, TBPc crosslinking was not detected, as expected (Figure 8B–D, and data not shown). As shown in Figure 8B–D, photocrosslinking between Mot1 and 5-IdU-substituted probes #1 and #3 was detected, whereas no crosslinking was detected to probes #2 and #5 (not shown). Among these probes, photocrosslinking of Mot1 to probes #1 and #3 was most robust, #4 and #7 less substantial, and #6 only slightly above background. The crosslinking of Mot1 to probes with 5-IdU substituted in only certain positions suggests that Mot1 occupies a specific position(s) in the ternary complex. Crosslinking to probes #1 and #3 was specific for 312 nm light, and was dependent on the presence of TBPc. While crosslinking was not significantly affected by the presence of ATP (Figure 8C), the absence of an ATP-dependent effect was expected because of the long irradiation time required for detection of crosslinked products and the fact that TBPc can rebind DNA following its displacement by Mot1 in the absence of a competitor (Auble and Hahn, 1993; Darst et al, 2003; Gumbs et al, 2003). Importantly, crosslinking of Mot1 to probes #1 and #4 shows that the protein contacts the major groove of the upstream DNA, consistent with the results shown in Figures 2 and 7. Crosslinking between Mot1 and probes #3 and #7 shows that Mot1 contacts the major groove of the TATA Box, suggesting that the TATA Box is sandwiched between the two proteins. Inhibition of DNA binding by preincubation of Mot1 and TBPc The unexpected interaction between Mot1 and TATA Box DNA is consistent with results suggesting an interaction between Mot1 and the TBP DNA-binding surface (Darst et al, 2001; Pereira et al, 2001; Klejman et al, 2005). Under conditions where TBP and TBPc bind rapidly and tightly to TATA Box bearing DNA (Figure 9, reaction scheme 1), detectable TATA Box occupancy was not observed when Mot1 was preincubated with these proteins (Figure 9, reaction scheme 2). Thus, Mot1 appears to wrap around the TBPc DNA-binding surface whether it is occupied or unoccupied by TATA DNA. Figure 8.Site-specific photocrosslinking of Mot1 to DNA. (A) DNA probes used. The native, unsubstituted sequence is shown. Asterisks indicate positions of 5-IdU substitution. (B) Radiolabeled DNAs (∼0.5 nM) were incubated with 8 nM TBPc±8 nM Mot1 and irradiated as indicated. Mot1 was added to all three reactions with unsubstituted DNA (‘no 5-IdU’) and as indicated for the others. Reactions were irradiated and products resolved on a 10% protein gel; crosslinked products were visualized by PhosphorImager analysis. There was no detectable crosslinking of Mot1 to probe nos. 2 and 5 (not shown). (C) Reactions were performed as in (B) with 312 nm UV light and the components as indicated. (D) Reactions were performed as in (B) with 312 nm UV irradiation for the times indicated. All reactions contained Mot1. In panels (B–D), arrow indicates the Mot1–DNA crosslinked product. Do
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