Selective Targeting and Inhibition of Yeast RNA Polymerase II by RNA Aptamers
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.27980
ISSN1083-351X
AutoresMuriel Thomas, Stéphane Chédin, Christophe Carles, Michel Riva, Michael Famulok, André Sentenac,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoTo probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II. A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation. Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. S. cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected. Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit. The selected RNA was expressed in yeast cells under the control of a Pol III promoter. Yeast cells that expressed the anti-Pol II aptamer grew normally. However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II. To probe the complex nucleic acid binding domains of yeast RNA polymerase II (Pol II), we have isolated in the presence of heparin RNA molecules that selectively bind to yeast Pol II. A class of RNA molecules was found to bind and strongly interfere with enzyme-DNA interaction but not with RNA chain elongation. Remarkably, one selected RNA ligand was a specific inhibitor of Saccharomyces cerevisiae Pol II. S. cerevisiae Pol I and Pol III and Pol II from Schizosaccharomyces pombe or wheat germ cells were not affected. Photocross-linking experiments showed that the RNA ligand preferentially interacted with B220, the largest subunit of Pol II and, to a lesser extent, with B150, the second largest subunit. The selected RNA was expressed in yeast cells under the control of a Pol III promoter. Yeast cells that expressed the anti-Pol II aptamer grew normally. However, a cell growth defect was observed when expressing the RNA aptamer in cells having an artificially reduced level of Pol II. During the transcription process, multisubunit RNA polymerases contact the DNA template and the RNA product at multiple sites to ensure the processivity of the polymerization reaction (Refs. 1Landick R. Roberts J.W. Science. 1996; 273: 202-203Crossref PubMed Scopus (9) Google Scholar and 2Markovtsov V. Mustaev A. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3221-3226Crossref PubMed Scopus (81) Google Scholarand references therein). All the subunits of bacterial RNA polymerase, including ς, participate in DNA binding (3Nudler E. Avetissova E. Markovtsov V. Goldfarb A. Science. 1996; 273: 211-217Crossref PubMed Scopus (172) Google Scholar, 4Dombroski A. Walter W. Gross C. Genes Dev. 1993; 7: 2446-2455Crossref PubMed Scopus (182) Google Scholar). Enzyme-DNA interaction is a dynamic process, and the extent of DNA protection does not remain invariant during RNA chain elongation (5Chan C. Landick R. Conaway R. Weliky Conaway J. Transcription Mechanisms and Regulation. 3. Raven Press, Ltd., New York1994: 297-322Google Scholar, 6Krummel B. Chamberlin M. J. Mol. Biol. 1992; 225: 239-250Crossref PubMed Scopus (92) Google Scholar). Furthermore, the enzyme plays an active role in holding the RNA product. Current models assume the existence of at least two separate RNA binding sites to account for the high stability and processivity of the transcription complexes as well as other particular features of the elongating RNA polymerase complexes (5Chan C. Landick R. Conaway R. Weliky Conaway J. Transcription Mechanisms and Regulation. 3. Raven Press, Ltd., New York1994: 297-322Google Scholar, 7Das A. Richardson C. Abelson J. Meister A. Walsh C. Annual Review of Biochemistry. 62. Annual Reviews Inc., Palo Alto, CA1993: 893-930Google Scholar, 8Chamberlin M. The Harvey Lectures. 88. Wiley-Liss, Inc., New York1995: 1-21Google Scholar). RNA polymerase-RNA interactions are functionally important at the various stages of the transcription reaction, yet our knowledge of the RNA binding sites is still very limited (2Markovtsov V. Mustaev A. Goldfarb A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3221-3226Crossref PubMed Scopus (81) Google Scholar, 9Liu K. Zhang Y. Severinov K. Das A. Hanna M.M. EMBO J. 1996; 15: 150-161Crossref PubMed Scopus (59) Google Scholar). RNA polymerase binds free RNA tightly (10Fox C.F. Gumport R. Weiss S.B. J. Biol. Chem. 1965; 240: 2101-2109Abstract Full Text PDF PubMed Google Scholar, 11Busby S. Spassky A. Buc H. Eur. J. Biochem. 1981; 118: 443-451Crossref PubMed Scopus (11) Google Scholar, 12Huaifeng M. Hartmann G. Eur. J. Biochem. 1983; 131: 113-118Crossref PubMed Scopus (12) Google Scholar), preferentially single-stranded RNA, and RNA binding induces the release of the ς factor from the bacterial holoenzyme as during promoter clearance, when the growing RNA chain reaches 8–10 nucleotides long (11Busby S. Spassky A. Buc H. Eur. J. Biochem. 1981; 118: 443-451Crossref PubMed Scopus (11) Google Scholar, 13Krakow J. von der Helm K. Transcription of Genetic Material. 35. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1970: 73-83Google Scholar). By filling the RNA product binding site, the nascent RNA causes a dramatic increase in the stability of the ternary complex (14Severinov K. Goldfarb A. J. Biol. Chem. 1994; 269: 31701-31705Abstract Full Text PDF PubMed Google Scholar, 15Levin J. Krummel B. Chamberlin M. J. Mol. Biol. 1987; 196: 85-100Crossref PubMed Scopus (126) Google Scholar), probably in relation with the conformation difference exhibited by the core (elongating) and holo (promoter binding) forms of bacterial RNA polymerase (16Polyakov A. Severinova E. Darst S.A. Cell. 1995; 83: 365-373Abstract Full Text PDF PubMed Scopus (151) Google Scholar). In addition, the product RNA plays an active role in pausing, termination, or anti-termination processes (5Chan C. Landick R. Conaway R. Weliky Conaway J. Transcription Mechanisms and Regulation. 3. Raven Press, Ltd., New York1994: 297-322Google Scholar, 17Johnson T.L. Chamberlin M.J. Cell. 1994; 77: 217-224Abstract Full Text PDF PubMed Scopus (64) Google Scholar, 18King R.A. Banik-Maiti S. Jin D.J. Weisberg R.A. Cell. 1996; 87: 893-903Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) by yet unclear mechanisms that probably involve interaction of the transcript with a site on elongating RNA polymerase. The functional importance of RNA-enzyme interactions prompted us to screen for RNA aptamers that bind to and inhibit yeast RNA polymerases at different steps of the transcription reaction. In the present work, we isolated high affinity RNA ligands against yeast RNA polymerase II (Pol II) 1The abbreviations used are: Pol, polymerase; bp, base pair; CTD, carboxyl-terminal domain; RPR, RNase P RNA. 1The abbreviations used are: Pol, polymerase; bp, base pair; CTD, carboxyl-terminal domain; RPR, RNase P RNA. using the Selex procedure (19Ellington A.D. Szostak J.W. Nature. 1990; 346: 818-822Crossref PubMed Scopus (7274) Google Scholar, 20Tuerk C. Gold L. Science. 1990; 249: 505-510Crossref PubMed Scopus (7689) Google Scholar) starting from a randomized RNA library. This strategy can yield RNA ligands that have a higher affinity for their protein target than the natural nucleic acid ligands (21Giver L. Bartel D. Zapp M. Pawul A. Green M. Ellington A. Nucleic Acids Res. 1993; 21: 5509-5516Crossref PubMed Scopus (114) Google Scholar, 22Breaker R.R. Banerjii A. Joyce G.F. Biochemistry. 1994; 33: 11980-11986Crossref PubMed Scopus (31) Google Scholar) or even bind nonnucleic acid-binding proteins (23Bock L.C. Griffin L.C. Latham J.A. Vermaas E.H. Toole J.J. Nature. 1992; 355: 564-566Crossref PubMed Scopus (2064) Google Scholar, 24Jellinek D. Lynott C.K. Rifkin D.B. Janjic N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11227-11231Crossref PubMed Scopus (146) Google Scholar, 25Jellinek D. Green L.S. Bell C. Janjic N. Biochemistry. 1994; 33: 10450-10456Crossref PubMed Scopus (159) Google Scholar, 26Lorsch J.R. Szostak J.W. Biochemistry. 1994; 33: 973-982Crossref PubMed Scopus (175) Google Scholar, 27Nieuwlandt D. Wecker M. Gold L. Biochemistry. 1995; 34: 5651-5659Crossref PubMed Scopus (112) Google Scholar). One selected RNA ligand described here specifically bound to Saccharomyces cerevisiae Pol II with high affinity and was a strong inhibitor of transcription. S. cerevisiaeRNA polymerases I, II, and III were prepared as described previously (28Buhler J.-M. Sentenac A. Fromageot P. J. Biol. Chem. 1974; 249: 5963-5970Abstract Full Text PDF PubMed Google Scholar, 29Dezélée S. Sentenac A. Fromageot P. FEBS lett. 1972; 21: 1-6Crossref PubMed Scopus (37) Google Scholar, 30Huet J. Riva M. Sentenac A. Fromageot P. J. Biol. Chem. 1985; 260: 15304-15310Abstract Full Text PDF PubMed Google Scholar). Schizosaccharomyces pombe Pol II was prepared analogously. Wheat germ Pol II was a generous gift from Dr. Dominique Job (Lyon). The initial random pool of RNAs was prepared as described (31Geiger A. Burgstaller P. von der Heltz H. Roeder A. Famulok M. Nucleic Acids Res. 1996; 24: 1029-1036Crossref PubMed Scopus (290) Google Scholar). DNA molecules of the library contained 40 randomized nucleotides flanked by 5′ and 3′ constant sequences recognized by primers. 5′ Primer, 5′-TCTAATACGACTCACTATAGGGCGCTAAGTCCTCGCTCA-3′; 3′ primer, 5′-GTCGGATCCGAGTCGCGCGT-3′. For the first round of selection, RNAs were produced by in vitro transcription of a DNA library fraction (10 μg of DNA, complexity of 1015 molecules (31Geiger A. Burgstaller P. von der Heltz H. Roeder A. Famulok M. Nucleic Acids Res. 1996; 24: 1029-1036Crossref PubMed Scopus (290) Google Scholar)) with T7 RNA polymerase (32Famulok M. J. Am. Chem. Soc. 1994; 116: 1698-1706Crossref Scopus (246) Google Scholar). RNAs were separated by electrophoresis on a 7.5% polyacrylamide, 7 m urea gel, and RNA molecules of appropriate length (approximately 80 nucleotides) were eluted from the gel as described by Johnson and Chamberlin (17Johnson T.L. Chamberlin M.J. Cell. 1994; 77: 217-224Abstract Full Text PDF PubMed Scopus (64) Google Scholar) and ethanol-precipitated. RNAs (40 pmol) were incubated with S. cerevisiae Pol II (5 pmol) for 30 min at 30 °C in 25 μl of binding buffer (50 mm Tris-HCl, pH 8, 100 mm ammonium sulfate). The RNA·RNA polymerase complexes were filtered under a slight vacuum through a HA filter (2.5 cm; 0.45 μm, Millipore) pre-wetted in binding buffer. The filter was rinsed with 6 ml of binding buffer, and the retained RNAs were extracted by phenol/urea treatment as described by Kubik et al. (33Kubik M.F. Stephens A.W. Schneider D. Marlar R.A. Tasset D. Nucleic Acids Res. 1994; 22: 2619-2626Crossref PubMed Scopus (117) Google Scholar). Selected RNAs were reverse-transcribed with the murine leukemia virus reverse transcriptase in the presence of the 3′ primer, and the cDNAs were amplified by polymerase chain reaction after the addition of the 5′ primer (Perkin-Elmer, Gene AmpR). cDNAs were used as templates for a next round of transcription/selection/amplification. Before the incubation with Pol II, RNAs were filtered on a HA filter to eliminate molecules nonspecifically retained on the filter. Every three rounds, reverse transcription-polymerase chain reaction products were purified by electrophoresis on a 7.5% polyacrylamide gel in denaturing conditions (see above). At the ninth round, cDNA (F9) were cloned and sequenced. Subsequently, six additional rounds of in vitroselection were performed under similar conditions, except that Pol II was preincubated with 250 ng of heparin (Sigma; H-2149 type) for 5 min at 30 °C in binding buffer before the addition of the RNAs. After the 15th round, cDNAs from the pool (F15) were cloned and sequenced. Standard incubation mixture (10 μl) contained 70 mm Tris-HCl, pH 8, 5 mmdithiothreitol, 75 mm ammonium sulfate, 2.5 mmMnCl2, 0.1 mm each ATP, CTP, GTP, UTP, 0.5 μCi of [α-32P]UTP (400 Ci/mmol), 2 pmol of dC-tailed template (37-bp double strand oligonucleotide containing a (dC)8 single strand extension at the 3′ extremity of the transcribed strand) or 1 μg of poly[d(A-T)]), 2.5 pmol of RNA polymerase, 1 unit of RNasin (Promega). For S. cerevisiaePol I, MnCl2 was replaced by MgCl2 (5 mm), whereas for Pol III both divalent cations were present. After a 30 min incubation at 30 °C, the amount of transcribed RNAs was quantified by precipitation in 5% (w/v) cold trichloroacetic acid and scintillation counting of precipitated radioactivity. The radiolabeled transcripts from the dC-tailed template were also analyzed by electrophoresis on a 20% (w/v) polyacrylamide, 7m urea gel and quantified with a PhosphorImager (Molecular Dynamics). The evaluation of apparentK d was performed by filter binding assays where 2 pmol of Pol II were incubated with varying amounts of FC- or FO-labeled RNAs (2 × 103 cpm/pmol). The binding reactions were filtered through a HA filter (2.5 cm, 0.45 μm; Millipore). After extensive washes with binding buffer, only bound RNAs were retained on filter. The radioactivity was then quantitated by scintillation counting. Apparent K d values were calculated using the Eadie-Hofstee plot: [RNA·Pol II] = [pol II] −K d [RNA·Pol II]/[RNA]. Pol II (0.8 pmol) was preincubated for 15 min at 30 °C with varying amounts of Pol II-specific aptamers in 15 μl of binding buffer. 5′-End-radiolabeled double-stranded dC-tailed template (see "RNA Polymerase Activity" above) (0.05 pmol, 105 cpm/pmol) was added, and incubation was continued for 15 min. After the addition of 2 μl of loading buffer (1 mm Tris-HCl, pH 8, 0.02% xylene cyanol, 0.02% bromphenol blue, 60% (w/v) sucrose), complexes were analyzed by electrophoresis on a 5% polyacrylamide gel and subjected to autoradiography. Pol II lacking CTD was prepared by digesting purified Pol II with proteinase K (enzyme/substrate = 1/10,000 (w/w)) during 30 min at 37 °C. After evaluation of the specific proteolysis of the CTD by SDS-polyacrylamide gel electrophoresis, the proteolyzed Pol II was chromatographed on a Mono Q column (Smart system, Pharmacia Biotech Inc.) to remove proteinase K. Nonspecific transcriptional activity of this enzyme was determined on calf thymus DNA and was found not to be affected by proteolysis. For characterizing the Pol II-DNA interactions, the transcribed or the nontranscribed strand of dC-tailed DNA template (see "RNA Polymerase Activity") were independently 5′-end-labeled using [γ-32P]ATP (Amersham) and T4 polynucleotide kinase (Pharmacia). After hybridization of each labeled strand with its nonlabeled complementary strand, free [γ-32P]ATP was eliminated by permeation chromatography on a Superdex 75 column in a 50 mm NaCl, 70 mmTris-HCl, pH 8, buffer on a Smart system (Pharmacia). For cross-linking experiments, 30 pmol of DNA template labeled on the coding or on the noncoding strand (5 × 104 cpm/pmol) were incubated with 6 pmol of proteolyzed or nonproteolyzed Pol II in a transcription buffer (4 mm dithiothreitol, 2.5 mmMnCl2, 30 mm Tris-HCl, pH 8, 130 mmammonium sulfate, and 0.1 mm each of ATP, CTP, GTP, UTP) for 60 min at 30 °C. Irradiation was performed on ice for 8 min at a distance of 0.5 cm using a 254-nm transilluminator (model VL-6LC, Bioblock). For characterizing the Pol II-FC interactions, FC RNA was body-labeled by in vitro transcription using T7 RNA polymerase and was purified by electrophoresis as described above. Pol II (2 pmol) was preincubated in the presence or the absence (see Fig. 6) of FO RNA (40 pmol) in binding buffer for 5 min at 30 °C in a final volume of 10 μl. Labeled FC RNA (4 pmol, 106 cpm) was added, and the incubation was pursued for 10 min. After irradiation, samples were supplemented with 20 μl of loading buffer and subjected to electrophoresis under denaturing conditions on a 7% polyacrylamide gel as described previously (34Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Proteins bands were visualized by silver staining (35Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3722) Google Scholar). The gel was dried and subjected to autoradiography on a Kodak Biomax film. Expression vector pIIIex426 RPR was a gift from Dr. Engelke (University of Michigan). This high multi-copy plasmid carried the URA3 auxotrophy marker, allowing selection of yeast cells growing on plates lacking uracil (36Good P.D. Engelke D.R. Gene. 1994; 151: 209-214Crossref PubMed Scopus (29) Google Scholar). FO1, FO2, and FC cDNA were prepared by reverse transcription-polymerase chain reaction using 5′ and 3′ oligonucleotide primers that carried EcoRI recognition sites and cloned at the EcoRI site of pIIIex426 RPR, yielding recombinant plasmids pIIIexFO1, pIIIexFO2, and PIIIexFC. FO1 and FO2 cDNAs were derived from the unselected pool, whereas FC cDNA corresponded to the selected FC ligand after round 15. Nucleotidic sequences and insertion sense were determined by sequencing (Pharmacia). Strain YF1971 (W303-1b with pLEU2-RPO21 LEU2) (37Archambault J. Jansma D.B. Friesen J.D. Genetics. 1996; 142: 737-747Crossref PubMed Google Scholar) was obtained by Dr. Friesen (University of Toronto). In this strain, the RPO21 gene that encodes the largest subunit of Pol II is placed under the control of a leucine-repressed promoter. Yeast transformation was done using the lithium acetate procedure. Yeast strains were grown at 30 °C on synthetic dextrose medium (38Sherman F. Methods Enzymol. 1991; 194: 3-20Crossref PubMed Scopus (2526) Google Scholar) supplemented or not with 0.2 mm leucine and isoleucine. RNA aptamers that bind to S. cerevisiae Pol II were selected by the Selex strategy. The starting RNA library (FO) consisted of a randomized region of 40 nucleotides flanked by defined sequences at the 5′- and 3′- ends (Fig. 1). An excess of RNAs (40 pmol) was incubated with a highly purified preparation of Pol II (5 pmol). The RNA·protein complexes were retained on a nitrocellulose filter by filtration, whereas free RNAs passed through the membrane. After extensive washing of the membrane, the protein·RNA complexes were dissociated by phenol extraction, and the RNA was amplified. This selection/amplification procedure was repeated nine times and resulted in the RNA pool (F9). Gel shift assays of increasing amounts of Pol II incubated with an identical amount of radiolabeled FO or F9 RNAs demonstrated that F9 RNAs exhibited a higher affinity for the enzyme compared with FO RNAs (data not shown). A fraction of F9 RNA molecules was reverse-transcribed, cloned, and sequenced. The sequence analysis of F9 molecules showed a nonrandom sequence distribution, but selected molecules were still heterogenous (data not shown). Therefore, additional selection/amplification cycles were performed using a more stringent screen. At each cycle, Pol II was preincubated with heparin before addition of the RNAs ligands. In this way, a competition was imposed between heparin and RNA for interaction with the nucleic acid binding sites of Pol II. By saturating Pol II with heparin, one would expect to select RNA ligands with a high affinity for nucleic acid binding sites or that were directed to alternative sites of the enzyme. After six rounds of high stringency selection, 34 clones of the final evolved population F15 were sequenced (Fig. 1). Based on sequence analysis, selected ligands were grouped into two classes: ligands of class 1 contained a conserved motif CGGN x GAGG ( x = 1 or 2, where N is any nucleotide), whereas no consensus motif was found in ligands of class 2 (Fig. 1). Among 27 ligands of class 1, three of them named FA, FB, and FC were found 13, 9, and 3 times, respectively. Within class 2, all of the RNA sequences were unique, except for FD, which was found twice (Fig. 1). F15 population bound Pol II with higher affinity and showed a greater capacity to inhibit transcription compared with the F9 and FO populations (data not shown). Therefore, we decided to investigate the properties of the F15 population by studying the monoclonal ligands FA, FB, FC, and FD, which were not unique in the final F15-evolved population. Individual clones of RNA ligands FA, FB, FC, and FD were assayed for their ability to inhibit Pol II activity in vitro using a 37-bp dC-tailed DNA template that could be used for transcription and DNA binding studies as well as for cross-linking experiments. Transcription of this short double-stranded DNA can be easily monitored by gel electrophoresis of the 37-mer RNA transcript. Before the transcription reaction, Pol II was preincubated with varying amounts of the initial RNAs pool (FO) or of the monoclonal FA, FB, FC, or FD ligands. As shown in Fig. 2, the preincubation of Pol II with the selected FA, FB, FC, or FD ligands inhibited transcription in a concentration-dependent manner, whereas at the same concentrations FO RNA pool had only a weak effect (Fig. 2). Note that the FD RNA, which does not contain the consensus motif previously defined, had the lower inhibitory effect. During the selection, the apparent K d of ligands for Pol II evolved from approximately 180 nm for FO to 20 nm for FC (data not shown). Compared with the other selected ligands, the FC RNA displayed the highest inhibitory potential. For this reason, FC was chosen for further investigations. Since the three forms of yeast RNA polymerase are highly homologous, in particular the two largest subunits, which represent two thirds of the mass of the enzymes, we analyzed the effect of the FC RNA on thein vitro transcriptional activity of Pol I, II, or III. Each enzyme was incubated with increasing amounts of FC RNA or FO pool and then assayed for their activity on a poly[d(A-T)] template. As previously observed with the dC-tailed template, Pol II activity was strongly inhibited by FC ligand at an equimolar ratio (Fig.3 A), whereas the FO pool had only a weak effect (10–20% inhibition; see Figs. 2 and3 B). Even when increasing the molar ratio FO/Pol II up to 6, FO RNA did not inhibit Pol II activity much (30% inhibition, data not shown). In the case of Pol I, a moderate inhibition of transcription was observed with a large amount of FC RNA (Fig. 3 A). A similar result was obtained in the presence of nonselected FO pool RNAs (Fig. 3 B), indicating that Pol I transcription shows a general, nonspecific sensitivity to RNA molecules. FC RNA or FO pool did not inhibit the Pol III activity but rather slightly activated the enzyme at sub-stoichiometric levels. These results indicated that the FC RNA was a specific inhibitor of the yeast Pol II. FC RNA was further examined for its ability to inhibit the activity of Pol II from various species. Before the transcription assay, each RNA polymerase was incubated with increasing amounts of FC RNA (Fig.4). In contrast to Pol II from S. cerevisiae, Pol II from S. pombe and wheat germ were not inhibited by the FC ligand. Thus, the FC ligand appeared to be a species-specific inhibitor of S. cerevisiae Pol II.Figure 4Species-specific FC inhibition. Pol II (2 pmol) from S. cerevisiae, S.pombe, or wheat germ was preincubated with FC RNA at different molar ratio as indicated. The enzyme activity was then assayed in standard conditions using a 37-bp dC-tailed DNA template. Acid-insoluble RNAs were quantified by counting in a scintillation counter. The enzyme activity is expressed in percent of activity of the enzyme without preincubation with FC RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We noted that the inhibition of Pol II transcription by FC RNA was independent of template concentration (from 0.5 to 3 μg per assay), suggesting that FC binding was noncompetitive with DNA (data not shown). To test whether FC RNA interfered with the binding of Pol II to a DNA template, we performed gel shift competition assays using the labeled dC-tailed template. Binding of purified Pol II to this probe led to the formation of two protein·DNA complexes of different electrophoretic mobility (Fig. 5, lane 2). The low mobility band resulted from the interaction of nonproteolyzed Pol II (retaining the carboxy-terminal domain or CTD) with DNA, whereas the higher mobility band contained the proteolyzed form of Pol II missing the CTD. Indeed, only the upper band could be shifted by an anti-CTD monoclonal antibody (data not shown). Preincubation of Pol II with FC ligand (Fig. 5, lanes 6–8) dramatically affected the binding of the labeled DNA probe to the enzyme. In the presence of an equimolar amount of FC RNA and Pol II, DNA binding of the probe to both enzyme forms was completely abolished. FC ligand not only prevented the binding of DNA but also displaced prebound template (data not shown). When exploring the effect of each selected RNA (FA, FB, FC, and FD) on the binding of Pol II on the DNA template using competition gel shift assays, we observed that in all cases the amount of DNA·Pol II complexes could be correlated to the inhibitory potency of the selected RNAs (data not shown). The differential effect of FC on Pol II activity as compared with Pol I and III (Fig. 3 A) was also confirmed by gel shift experiments, since a 2-fold molar excess of FC RNA was unable to inhibit the formation of Pol I·DNA or Pol III·DNA complexes (data not shown). In contrast, the FO RNA pool only partially decreased enzyme-DNA binding when added in a 2-fold 2-fold molar excess relative to the Pol II (Fig. 5, lane 5). Surprisingly, FO RNA appears to diminish binding of the DNA template (Fig. 5, comparelanes 3, 4, and 5 with lane 2) at FO RNA/Pol II ratios that have almost no effect on Pol II transcriptional activity (see Fig. 2). The apparent discrepancy between binding assay and Pol II activity assay could be due to the presence of a nonactive subpopulation of Pol II in the enzyme preparation. Although experimental data supporting this hypothesis are lacking, FO RNA could possibly inhibit DNA binding by the transcriptionally incompetent enzyme population. Using competition binding assays, we observed that the affinity of FC for Pol II did not depend on temperature (from 0 to 30 °C) or ammonium sulfate concentration (from 0 to 100 mm) (data not shown). To test whether the single-stranded DNA version of FC (FC DNA) displayed inhibitory properties as FC RNA, gel shift experiments as well as transcription assays were performed in the presence of FC DNA produced by asymetric polymerase chain reaction. In contrast to FC RNA, a 4-fold molar excess of FC DNA relative to the Pol II only partially impaired the dC-tailed DNA·pol II complex formation and did not inhibit the pol II-dependent transcription (data not shown). To identify the polymerase subunit(s) contacted by FC RNA ligand, Pol II was cross-linked to 32P-labeled FC RNA by UV irradiation, and the labeled subunits were identified after SDS-polyacrylamide gel electrophoresis and autoradiography (Fig.6 A). In a first experiment, under electrophoretic conditions at which all Pol II subunits could be visualized, we found that only the largest subunits were detectably labeled (data not shown). Fig. 6 A, lane 1 shows the two largest subunits (B220 and B150) of Pol II resolved on a 7.5% SDS-polyacrylamide gel electrophoresis. We determined by immunoblot analysis that the major intermediary B185 component derived from B220 by proteolysis of the CTD and the minor products ranging from 160 to 185 kDa (indicated by a vertical line) corresponded to further proteolysis of B220 (data not shown). Most of the FC RNA covalently bound to Pol II reflected a preferential cross-linking to the B220 and B185 subunits and suggested that the CTD is not involved in this interaction (Fig. 6 A, lane 2). The weakly labeled bands that migrated faster than B185 were derived from the cross-linkage of B220 derivatives (vertical line) or B150. In a control experiment, no radioactive band was detected upon UV-irradiation of the FC RNA in the absence of RNA polymerase (Fig.6 A, lane 4). Furthermore, the preincubation of Pol II with a large excess of nonradioactive FO RNA pool did not reduce the labeling of the subunits targeted by FC RNA (Fig. 6 A,lane 3), hence confirming the specificity of the binding and cross-linking reactions. We next compared the Pol II-binding sites of the FC RNA ligand with those of a DNA template, since the FC RNA competed for DNA binding. This was achieved by cross-linking of Pol II to a32P-labeled dC-tailed DNA template. Analysis of cross-linked subunits was performed as before (Fig. 6 B). As previously mentioned, CTD is not involved in the interaction of FC RNA with Pol II. Nevertheless, Suzuki (39Suzuki M. Nature. 1990; 344: 562-565Crossref PubMed Scopus (90) Google Scholar) demonstrated that a synthetic peptide corresponding to the CTD could bind in vitro to DNA. To check the involvement of the CTD in the binding of Pol II to DNA, two Pol II preparations were used for the characterization of the Pol II-DNA template interactions: one essentially containing the intact B220 subunit, the other containing the proteolyzed B185 form. Enzyme-DNA UV photocross-linking was performed using a DNA template selectively labeled either in the transcribed or the nontranscribed strand. We first determined that among all subunits of Pol II, only the two largest, B220 and B150, were cross-linked to the template (data not shown) as well as B185, the form of B220 lacking the CTD. Comparison of the relative amount of the B220·DNA and B185·DNA complexes (Fig.6 B, compare lanes 5 with 7 or6 with 8) revealed that the CTD was not involved in the interaction of B220 subunit with the DNA strands. Note that the largest subunit was cross-linked to the same extent to the transcribed and the nontranscribed strands, whereas the B150 subunit was predominantly cross-linked to the non-transcr
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