Rrn3 Becomes Inactivated in the Process of Ribosomal DNA Transcription
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m301093200
ISSN1083-351X
AutoresIwona Hirschler‐Laszkiewicz, Alice Cavanaugh, Ayoub Mirza, Mingyue Lun, Qiyue Hu, Tom Smink, Lawrence I. Rothblum,
Tópico(s)RNA Research and Splicing
ResumoThe human homologue of yeast Rrn3, a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription. Although the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined. It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I·Rrn3 complexes that can interact with the committed template. These and other reported differences would have implications with respect to the mechanism by which Rrn3 functions in transcription. For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically. Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically. We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed. Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions. For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction. We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I. Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription and that its ability to associate with RNA polymerase I is lost upon transcription. The human homologue of yeast Rrn3, a 72-kDa protein, is essential for ribosomal DNA (rDNA) transcription. Although the importance of Rrn3 function in rDNA transcription is well established, its mechanism of action has not been determined. It has been suggested that the phosphorylation of either yeast RNA polymerase I or mammalian Rrn3 regulates the formation of RNA polymerase I·Rrn3 complexes that can interact with the committed template. These and other reported differences would have implications with respect to the mechanism by which Rrn3 functions in transcription. For example, in the yeast rDNA transcription system, Rrn3 might function catalytically, but in the mammalian system it might function stoichiometrically. Thus, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically. We report that mammalian Rrn3 becomes the limiting factor as transcription reactions proceed. Moreover, we demonstrate that Rrn3 is inactivated during the transcription reactions. For example, Rrn3 isolated from a reaction that had undergone transcription cannot activate transcription in a subsequent reaction. We also show that this inactivated Rrn3 not only dissociates from RNA polymerase I, but is not capable of forming a stable complex with RNA polymerase I. Our results indicate that Rrn3 functions stoichiometrically in rDNA transcription and that its ability to associate with RNA polymerase I is lost upon transcription. Three requirements must be met before RNA polymerase I (pol I) 1The abbreviations used are: pol I, RNA polymerase I; rDNA, ribosomal DNA; TBP, TATA-binding protein; TAF, TBP-associated factors; UBF, upstream binding factor; nt, nucleotide; CHX, cycloheximide.1The abbreviations used are: pol I, RNA polymerase I; rDNA, ribosomal DNA; TBP, TATA-binding protein; TAF, TBP-associated factors; UBF, upstream binding factor; nt, nucleotide; CHX, cycloheximide. can initiate specific and effective transcription. Both the ribosomal DNA (rDNA) promoter and RNA polymerase I have to be “transcription ready,” and they must join together to form a functional initiation complex. The assembly of the intermediate and final complexes involved in this process requires a complex series of protein-DNA and protein-protein interactions. For example, the stable binding of the transcription factors to the rDNA promoter requires the coordinate binding of factors to the core and upstream promoter elements (1Paule M.R. AAAATranscription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I. Springer-Verlag, New York1998Google Scholar).Eukaryotic rDNA promoters contain a core promoter element and an upstream promoter element (for review, see Ref. 1Paule M.R. AAAATranscription of Ribosomal RNA Genes by Eukaryotic RNA Polymerase I. Springer-Verlag, New York1998Google Scholar). Two multi-subunit complexes, core factor (CF) and upstream activating factor (UAF), which bind to the core promoter and to the upstream element, respectively (2Learned R.M. Cordes S. Tjian R. Mol. Cell. Biol. 1985; 5: 1358-1369Crossref PubMed Scopus (122) Google Scholar, 3Keys D.A. Vu L. Steffan J.S. Dodd J.A. Yamamoto R.T. Nogi Y. Nomura M. Genes Dev. 1994; 8: 2349-2362Crossref PubMed Scopus (77) Google Scholar) are required to commit the yeast rDNA promoter. Both CF and UAF interact specifically with TATA-binding protein (TBP) (4Keys D.A. Lee B.S. Dodd J.A. Nguyen T.T. Vu L. Fantino E. Burson L.M. Nogi Y. Nomura M. Genes Dev. 1996; 10: 887-903Crossref PubMed Scopus (115) Google Scholar, 5Steffan J. Keys D. Dodd J. Nomura M. Genes Dev. 1996; 10: 2551-2563Crossref PubMed Scopus (77) Google Scholar). In mammals, two known transcriptional factors are required to commit rDNA promoter. The binding of selectivity factor (SL1) (6Lin C.W. Moorefield B. Payne J. Aprikian P. Mitomo K. Reeder R.H. Mol. Cell. Biol. 1998; 16: 6436-6443Crossref Scopus (60) Google Scholar, 7Comai L. Tanese N. Tjian R. Cell. 1992; 68: 965-976Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 8Zomerdijk J.C. Beckmann H. Comai L. Tjian R. Science. 1994; 266: 2015-2018Crossref PubMed Scopus (90) Google Scholar, 9Comai L. Zomerdijk J.C. Beckmann H. 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Both SL1 and UBF are subject to regulation via phosphorylation and acetylation (14O'Mahony D.J. Smith S.D. Xie W.Q. Rothblum L.I. Nucleic Acids Res. 1992; 20: 1301-1308Crossref PubMed Scopus (61) Google Scholar, 15O'Mahony D.J. Xie W.Q. Smith S.D. Singer H.A. Rothblum L.I. J. Biol. Chem. 1992; 267: 35-38Abstract Full Text PDF PubMed Google Scholar, 16Heix J. Vente A. Voit R. Budde A. Michaelidis T.M. Grummt I. EMBO J. 1998; 17: 7373-7381Crossref PubMed Scopus (132) Google Scholar, 17Pelletier G. Stefanovsky V.Y. Faubladier M. Hirschler-Laszkiewicz I. Savard J. Rothblum L.I. Cote J. Moss T. Mol. Cell. 2000; 6: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 18Hirschler-Laszkiewicz I. Cavanaugh A. Hu Q. Catania J. Avantaggiati M.L. Rothblum L.I. Nucleic Acids Res. 2001; 29: 4114-4124Crossref PubMed Scopus (60) Google Scholar, 19Grummt I. Voit R. EMBO J. 2001; 20: 1353-1362Crossref PubMed Scopus (175) Google Scholar). In addition, Rb, the protein product of the retinoblastoma susceptibility gene, interacts with UBF repressing pol I transcription (20Cavanaugh A.H. Hempel W.M. Taylor L.J. Rogalsky V. Todorov G. Rothblum L.I. Nature. 1995; 374: 177-180Crossref PubMed Scopus (290) Google Scholar), and SV40 large T antigen activates pol I transcription by interacting with SL1 (21Zhai W.G. Tuan J.A. Comai L. Genes Dev. 1997; 11: 1605-1617Crossref PubMed Scopus (48) Google Scholar).The mechanism whereby RNA pol I is recruited to the promoter is unclear. It was established (22Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (117) Google Scholar) that only ∼2% of RNA pol I population present in an exponentially growing yeast cell is capable of promoter-specific transcription. These competent RNA pol I molecules were found to contain core RNA polymerase I subunits and Rrn3, a polymerase-associated factor.Both genetic and biochemical experiments demonstrate that yeast Rrn3 is essential for rDNA transcription. The human homologue has been cloned (23Moorefield B. Greene E.A. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4724-4729Crossref PubMed Scopus (85) Google Scholar) and subsequently identified as the previously described transcription initiation factor IA (TIF-IA) (24Bodem J. Dobreva G. Hoffmann-Rohrer U. Iben S. Zentgraf H. Delius H. Vingron M. Grummt I. EMBO Rep. 2000; 1: 171-175Crossref PubMed Scopus (112) Google Scholar). Current models suggest that Rrn3 acts as a bridge between RNA pol I and the committed rDNA promoter (25Yamamoto R. Nogi Y. Dodd J. Nomura M. EMBO J. 1996; 15: 3964-3973Crossref PubMed Scopus (102) Google Scholar, 26Thuriaux P. Mariotte S. Buhler J.M. Sentenac A. Vu L. Lee B.S. Nomura M. J. Biol. Chem. 1995; 270: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 27Peyroche G. Milkereit P. Bischler N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (138) Google Scholar, 28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 29Miller G. Panov K.I. Friedrich J.K. Trinkle-Mulcahy L. Lamond A.I. Zomerdijk J.C. EMBO J. 2001; 20: 1373-1382Crossref PubMed Scopus (146) Google Scholar). A direct interaction between the 43-kDa subunit of pol I (rpa43) and Rrn3 in the Rrn3·pol I complex was confirmed (27Peyroche G. Milkereit P. Bischler N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (138) Google Scholar, 28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) as well as the direct interaction of human Rrn3 with the TAFI110 and TAFI63 subunits of species-specific transcription factor SL1 (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 29Miller G. Panov K.I. Friedrich J.K. Trinkle-Mulcahy L. Lamond A.I. Zomerdijk J.C. EMBO J. 2001; 20: 1373-1382Crossref PubMed Scopus (146) Google Scholar). Despite this body of knowledge, there are still significant controversies concerning the role that Rrn3 plays in transcription.Fath et al. (30Fath S. Milkereit P. Peyroche G. Riva M. Carles C. Tschochner H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14334-14339Crossref PubMed Scopus (61) Google Scholar) proposed that the phosphorylation of yeast RNA pol I regulates the formation of a functional initiation complex between RNA pol I and Rrn3. In contrast, our recently published data (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) indicate that the phosphorylation state of mammalian Rrn3 regulates this process. Aprikian et al. (31Aprikian P. Moorefield B. Reeder R.H. Mol. Cell. Biol. 2001; 21: 4847-4855Crossref PubMed Scopus (55) Google Scholar) reported that yeast RNA pol I could be recruited to the rDNA promoter in the absence of Rrn3. However, they noted that these complexes could not be converted to transcriptionally active complexes when Rrn3 was added to the reactions. In contrast Schnapp et al., (32Schnapp A. Schnapp G. Erny B. Grummt I. Mol. Cell. Biol. 1993; 13: 6723-6732Crossref PubMed Scopus (69) Google Scholar) reported that mammalian “TIF-IA (Rrn3) is liberated from the initiation complex and facilitates transcription from templates bearing preinitiation complexes which lack TIF-IA.” This observation is significantly different from the Aprikian model. In addition, it suggests that Rrn3 functions catalytically. This is in contrast to earlier observations that factor C*, a factor that shares many properties with Rrn3, functioned stoichiometrically (33Brun R.P. Ryan K. Sollner-Webb B. Mol. Cell. Biol. 1994; 14: 5010-5021Crossref PubMed Scopus (35) Google Scholar). As these differences would have significant implications with respect to the mechanism by which Rrn3 functions in transcription, we examined the question as to whether Rrn3 functions catalytically or stoichiometrically.A catalyst is defined as an element that modifies the rate of a chemical reaction without being consumed in the process, and without being changed by the consequences of the process. Rrn3 was shown (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) to undergo modifications that are possibly the consequence of transcription. In this study we present evidence that Rrn3 is consumed during transcription and cannot be directly “reused” in a second round of transcription. We also demonstrate that Rrn3 dissociates from pol I as a consequence of transcription. These observations provide evidence that Rrn3 serves a stoichiometric rather then catalytic function in RNA pol I transcription. This in turn supports our hypothesis that the post-translational modification of Rrn3 plays an active role in the formation of the functional Rrn3·pol I complex in mammalian cells.EXPERIMENTAL PROCEDURESCell Culture and Treatment with Cycloheximide—N1S1 cells were grown in RPMI1640 + 5% fetal bovine serum (34Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Where indicated, cells were treated with cycloheximide, 2 μg/ml (Sigma) for one hour.Production of Recombinant rpa43 and Rrn3 in Sf9 Cells and Protein Purification—S, FLAG-tagged Rrn3 and S, His rpa43 were expressed in Sf9 cells and purified using anti-FLAG agarose beads (Sigma) or nickel-nitrilotriacetic acid-agarose (Qiagen) as previously described (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To recover recombinant FLAG-tagged Rrn3 from the in vitro transcription reactions, the 200-μl reactions were diluted 2.5-fold by adding 300 μl of transcription buffer containing 20 mm HEPES, pH 7.9, 100 mm KCl, 5 mm MgCl2, 0.2 mm EDTA, and 6% glycerol. The NaCl concentration was then adjusted to a final concentration of 150 mm. Both changes were necessary for the optimal binding of Rrn3 to anti-FLAG beads. 10 μl of packed anti-FLAG agarose beads were added to each reaction, and the reaction tubes were tumbled for two hours at 4 °C. After the beads were washed three times with 1 ml of wash buffer (50 mm Tris-HCl, pH 7.9, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100), Rrn3 was eluted with 30 μl of 0.5 mg/ml FLAG peptide (Sigma). The protein was used immediately for the second round of transcription, Western blot analysis, or co-immunoprecipitation with rpa43.Purification of UBF and SL—UBF and SL1 were purified by DEAE Sephadex and heparin agarose chromatography as described previously (35Smith S. Oriahi E. Lowe D. Yang-Yen H.F. O'Mahony D. Rose K. Chen C. Rothblum L.I. Mol. Cell. Biol. 1990; 10: 3105-3116Crossref PubMed Scopus (75) Google Scholar). UBF was eluted from the heparin agarose column with 600 mm KCl, whereas SL-1 was eluted with 1000 mm KCl. At these stages of purification there is no detectable Rrn3 or pol I in either the UBF or SL-1 preparations (data not shown).Co-immunoprecipitation of Rrn3/RNA Pol I—Rrn3 that had been incubated in a transcription reaction was then incubated with anti-FLAG beads and eluted with FLAG peptide. 30 μl of eluted Rrn3 (∼7 μg) was mixed with 100 μl(∼100 μg) of recombinant rpa43 and tumbled at 4 °C overnight in a final volume of 100 μl of buffer containing 20 mm HEPES, pH 7.9, 100 mm KCl, 5 mm MgCl2, 0.2 mm EDTA, and 6% glycerol. Anti-FLAG agarose beads were added and the mixture tumbled for two hours at 4 °C. The beads were then washed as described above and Rrn3 eluted with FLAG peptide. The protein was used immediately for Western blot analysis.rDNA Transcription Templates—pU5.1E/X contains the rat 45 S rDNA (-286 to + 630) promoter and pUE/B is a PCR product of the rat 45 S rDNA (-279 to + 480) promoter. When truncated with EcoRI the transcript from pU5.1E/X is 632, and that from pUE/B is 480 nts (35Smith S. Oriahi E. Lowe D. Yang-Yen H.F. O'Mahony D. Rose K. Chen C. Rothblum L.I. Mol. Cell. Biol. 1990; 10: 3105-3116Crossref PubMed Scopus (75) Google Scholar).Protein Extracts—S-100, whole cell extracts from control or cycloheximide-treated N1S1 cells and nuclear protein extracts from rat hepatoma cells were prepared essentially as described (34Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 36Haglund R.E. Rothblum L.I. Mol. Cell. Biochem. 1987; 73: 11-25Crossref PubMed Scopus (27) Google Scholar).In Vitro Transcription—In vitro transcription reactions were carried as described previously (37Gokal P.K. Cavanaugh A.H. Thompson E.A. J. Biol. Chem. 1986; 261: 2536-2541Abstract Full Text PDF PubMed Google Scholar, 38Cavanaugh A.H. Gokal P.K. Lawther R.P. Thompson E.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 718-721Crossref PubMed Scopus (33) Google Scholar).Western Blot Analysis—SDS-PAGE and electroblot analysis were performed as described previously (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Polyclonal rabbit antisera to the 194-kDa (β′) and 127-kDa (β) subunits of pol I have been described previously (34Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Polyclonal, rabbit antisera to Rrn3 and rpa43 were raised to purified, recombinant Rrn3 or rpa43, respectively. S peptide conjugated to horseradish peroxidase (S-HRP conjugate, Novagen) was used as recommended by the suppliers. Monoclonal anti-FLAG antibodies were used as recommended by the supplier (Sigma).RESULTSCycloheximide Inactivates Rrn3—We have previously reported that whole cell extracts prepared from CHX-treated cells cannot support transcription in vitro and that transcription activity can be restored by addition of Rrn3 (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, our evidence is consistent with the model that treatment with cycloheximide results in the inactivation of mammalian Rrn3. However, it is possible that cycloheximide inactivates rDNA transcription through another pathway, and the rescue of rDNA transcription by exogenous Rrn3 is due to a redundancy in the mechanism of rDNA transcription, i.e. Rrn3 can replace another component. To test this model we isolated Rrn3 from cells treated with cycloheximide and from control cells (Fig. 1A). 3T3 cells were transfected with pCDNA3.1FLAG-Rrn3 as described previously (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Twenty-four hours later, one half of the cells were treated with cycloheximide for one hour. Rrn3 was purified from whole cell extracts of both groups of cells by immuno-affinity purification using immobilized anti-FLAG antibodies (28Cavanaugh A.H. Hirschler-Laszkiewicz I. Hu Q. Dundr M. Smink T. Misteli T. Rothblum L.I. J. Biol. Chem. 2002; 277: 27423-27432Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Equal amounts of the two forms of Rrn3 were added to extracts from cycloheximide-treated cells to determine whether they were equally active in transcription (Fig. 1B). The observation that Rrn3 purified from cells treated with cycloheximide (lane 3) was <5% as active as the Rrn3 purified from untreated cells (lane 4) provided additional evidence that treatment with cycloheximide resulted in the inhibition of Rrn3 activity. Moreover, when this result is considered with other observations, it is consistent with the model that the only component of the rDNA transcription apparatus that is inactivated in extracts from cycloheximide-treated cells is Rrn3.Rrn3 Is the Limiting Factor in in Vitro Transcription Assays—The question as to whether Rrn3 functions catalytically or stoichiometrically has been examined indirectly (32Schnapp A. Schnapp G. Erny B. Grummt I. Mol. Cell. Biol. 1993; 13: 6723-6732Crossref PubMed Scopus (69) Google Scholar), and those authors reported data consistent with a model in which Rrn3 functions catalytically. As this question has a significant effect on our understanding of the mechanisms involved in transcription initiation we examined this question using three different approaches.Our first series of experiments were based on the premise that if Rrn3 undergoes some inactivation as the result of transcription, it should become limiting in the transcription reaction as time proceeds and transcription should plateau. In turn, transcription should resume when the reaction was supplemented with Rrn3 (Fig. 2A). In a typical transcription reaction, the accumulation of transcription products reached a plateau by 30 min (Fig. 2B, lanes 1–5). If Rrn3 was inactivated during transcription, then the addition of Rrn3 should cause transcription to begin again. As shown, the addition of Rrn3 to a transcription reaction, which had been incubated for 60 min, stimulated transcription (Fig. 2B, compare lanes 6, 7, and 8 with lane 3). In a second set of experiments (Fig. 2C), we compared the levels of transcript accumulated in reactions that were supplemented with Rrn3 after 60 min of transcription to the amount that might accumulate if Rrn3 was not added. The levels of transcript that accumulated under those conditions were compared with the levels that accumulated in standard reactions that had run for 30 or 60 min (lanes 1 and 2) in the presence of [α32-P]UTP. The second group of reactions was allowed to proceed for 60 min in the absence of radioactive probe (lanes 3 and 4). After 60 min of transcription, [α32-P]UTP was added to both reactions, and transcription was allowed to proceed for an additional 30 min. Rrn3 was added to only one reaction (lane 4). When the transcription reactions were complete, the in vitro synthesized RNA was isolated, fractionated by urea-PAGE, and detected by autoradiography. The absence of transcript in lane 3 suggests that an essential component(s) was consumed during the first 60 min of reaction. The observation that transcripts accumulated when the reaction was supplemented with Rrn3 at the same time as isotope was added (lane 4), suggests that Rrrn3 was the “consumed” component. Taken together the results shown in Fig. 2 demonstrate that Rrn3 is inactivated or consumed as a result of transcription.Fig. 2Rrn3 becomes a limiting factor during transcription in vitro.A, schematic depicting the theoretical result of adding recombinant Rrn3 to a transcription reaction after 60 min, assuming that Rrn3 has become rate-limiting. B, transcription reactions, prepared as described under “Experimental Procedures,” using 5 μl of control N1S1 S-100 extract, to which recombinant Rrn3 was added as indicated. Lanes 1–5 show a time course without addition of Rrn3. Reaction time was 5, 30, 60, 90, and 120 min. Lanes 6–8 show the reactions that were run for additional 15, 30, or 60 min after adding 100 ng of Rrn3 at 60 min. C, transcription reactions were performed using 5 μl of cycloheximide-treated S-100 N1S1 protein extract, 1.5 ng of recombinant Rrn3 and 0.1 μg template pU5.1E/X. Lanes 1 and 2 show reactions run for 30 and 60 min. Lanes 3 and 4 show reactions that were run for 60 min without isotope. Isotope was added at 60 min along with 0.5 ng Rrn3 (lane 4) or without Rrn3 (lane 3), and transcription was allowed to proceed for another 15 min. Transcription reactions, purification of the in vitro synthesized RNA, and PAGE analysis of the transcription reactions were carried out as described under “Experimental Procedures.” Trans., the 632-nt transcript that results from correct initiation. Int. Std., internal standard added for the recovery of nucleic acids.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Template Commitment Assay—Our second approach was to use a modified template commitment assay (Fig. 3). Two templates, distinguished by the sizes of the transcripts they support, were used in these assays (template 1, p5.1E/X, 632 nt transcript; and template 2, pUE/B, 480 nt transcript). The first experiments used extracts from cycloheximide-treated cells as the use of unfractionated extracts avoids artifacts associated with purification and fractionation. As shown in Fig. 3, if both templates are added at the same time, both are transcribed (lane 1). However, if the reaction is incubated with only one template followed by the addition of the second template after 60 min (followed in turn by an additional 30 min of transcription), transcription is only observed from the template that was incubated with Rrn3 first (template 1, lanes 2 and 3). When the second template 2 was added together with Rrn3, again both templates are transcribed (lane 4). These data suggest that, under these conditions, Rrn3 mediates an essential transcription step and is the limiting component in the transcription reaction.Fig. 3Rrn3 commits to the template.Upper panel, a schematic depicting the experimental protocol. Lower panel, once Rrn3 is preincubated with one template it does not support transcription from a second template (lanes 2 and 3). Template 2 was added to the reaction either at the same time as template 1 (lane 1) or 60 min after reaction was started (lanes 2–4). Template 2 was added in the mixture containing additional 7 ng Rrn3 (lane 4) or without Rrn3 (lanes 1, 2, and 3). All assays contained 7 ng of FLAG-tagged Rrn3 and 5 μl of S-100 extract from CHX-treated cells. Trans. 1, the 632-nt transcript that results from the correct initiation on template 1. Trans. 2, the 480-nt transcript from template 2. Int. Std., internal standard added for the recovery of nucleic acids.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To ensure that Rrn3 was the limiting component in the transcription reactions, a similar experiment was carried out, and the reactions were supplemented with purified UBF and SL1, so that those factors would not be limiting (Fig. 4). When purified, Rrn3, SL1, and UBF were added to an in vitro reaction mixture containing S-100 from CHX-treated N1S1 (as a source of RNA pol I) and either template 1 or 2, each template was transcribed (lanes 2 and 3 and 5 and 6). If both templates (Temp 1 and 2) were added at time 0, both templates are transcribed (lane 7). However, if the second template mixture containing SL1 and UBF is added after 20, 40, or 60 min, only the first (Temp 1) template is transcribed (lanes 8–10). As SL1 and UBF were added along with the second template, they are not limiting factors in these reactions. This suggests that once Rrn3 commits to the initiation complex, it cannot contribute to transcription from a second template.Fig. 4Rrn3, and not SL1 and UBF, determines the formation of the preinitiation complex.Upper panel, a schematic depicting the experimental protocol. Lower panel, in vitro transcription assays were performed using FLAG-tagged Rrn3, 5 μl CHX S-100 extract, and 10 μl of each chromatography fraction HS 600 (purified UBF), and HS 1000 (purified SL1). Template 1 (100 ng) was added to the reaction either at the same time as template 2 (30 ng) (lane 7) or at varying times after transcription was started (20, 40, or 60 min, lanes 8–10, respectively), and transcription was allowed to proceed for an additional 30 min for a total of 50, 70, and 90 min, respectively. Template 1 was added in the mixture containing an additional 10 μl each of UBF and SL1 fraction. Lanes 2, 3, 5, and 6 show each template transcribed separately. Transcription reactions, purification of the in vitro synthesized RNA, and PAGE analysis of the transcription reactions were carried out as described under “Experimental Procedures.” Trans. 1, the 480-nt transcript that results from the correct initiation on template 1. Trans. 2, the 632-nt transcript from template 2. Int. Std., internal standard added for the recovery of nucleic acids.View Large Image Figure ViewerDownload Hi-res image
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