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• W2088808837 abstract "Article1 July 1998free access A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription Philipp Milkereit Philipp Milkereit BZH Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany Search for more papers by this author Herbert Tschochner Corresponding Author Herbert Tschochner BZH Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany Search for more papers by this author Philipp Milkereit Philipp Milkereit BZH Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany Search for more papers by this author Herbert Tschochner Corresponding Author Herbert Tschochner BZH Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany Search for more papers by this author Author Information Philipp Milkereit1 and Herbert Tschochner 1 1BZH Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3692-3703https://doi.org/10.1093/emboj/17.13.3692 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Only a small proportion (<2%) of RNA polymerase I (pol I) from whole-cell extracts appeared to be competent for specific initiation at the ribosomal gene promoter in a yeast reconstituted transcription system. Initiation-competent pol I molecules were found exclusively in salt-resistant complexes that contain the pol I-specific initiation factor Rrn3p. Levels of initiation-competent complexes in extracts were independent of total Rrn3p content and varied with the growth state of the cells. Although extracts from stationary phase cells contained substantial amounts of Rrn3p and pol I, they lacked the pol I–Rrn3p complex and were inactive in promoter-dependent transcription. Activity was restored by adding purified pol I–Rrn3p complex to extracts from stationary phase cells. The pol I–Rrn3p complex dissociated during transcription and lost its capacity for subsequent reinitiation in vitro, suggesting a stoichiometric rather than a catalytic activity in initiation. We propose that the formation and disruption of the pol I–Rrn3p complex reflects a molecular switch for regulating rRNA synthesis and its growth rate-dependent regulation. Introduction Promoter utilization by eukaryotic RNA polymerases requires accessory proteins termed general transcription factors or initiation factors. Even in the presence of saturating amounts of initiation factors, however, purified RNA polymerases typically exhibit low efficiencies of template utilization. For instance, homogenous RNA polymerase II from Saccharomyces cerevisae synthesized <10 specific transcripts per hundred DNA template molecules under optimal conditions with purified general factors (Sayre et al., 1992a,b). This matched the maximum efficiency observed in nuclear extract (Chasman et al., 1989), indicating that low template utilization was not due to loss of activity during purification of transcription proteins. One possible explanation for this apparent inefficiency is that only a fraction of eukaryotic RNA polymerases are active for transcription initiation at any one time. For instance, RNA polymerase II in vivo exists in various forms distinguished by the phosphorylation state of the C-terminal repeat domain (CTD) in the largest subunit (reviewed in Dahmus, 1994). Only the dephosphorylated form is thought to be competent for assembly into ‘pre-initiation complexes’ with accessory proteins on promoter DNA. Furthermore, distinct activated and non-activated RNA polymerase II complexes have been described recently in yeast (Akhtar et al., 1996). Biochemical analyses of cell-free transcription systems for RNA polymerase I (pol I) from Acanthamoeba (Stevens and Pachler, 1973; Bateman and Paule, 1986), mouse (Tower and Sollner Webb, 1987) and yeast (Milkereit et al., 1997) have identified at least two different forms of polymerase, only one of which is able to initiate at the rDNA promoter. Neither the relative proportions of pol I in each form nor the underlying molecular mechanisms for establishing and/or interconverting them have been elucidated. Several reports implicate these distinct forms of pol I in growth rate-dependent regulation of rRNA synthesis. Homogeneous or enriched pol I that supports promoter-specific transcription has been isolated exclusively from logarithmically growing cells. This active form of the enzyme can complement inactive extracts from stationary phase cells in systems from Acanthamoeba (Paule, 1983; Bateman and Paule, 1986), mouse (Tower and Sollner Webb, 1987) and yeast (Riggs et al., 1995). More detailed analyses of the mouse system correlate growth-dependent initiation activity with a transcription factor that binds tightly to pol I. This factor, variously termed TIFIA (Buttgereit et al., 1985; Schnapp et al., 1990, 1993), Factor C* (Brun et al., 1994) and TFIC (Gokal et al., 1990; Mahajan and Thompson, 1990; Mahajan et al., 1990), can be separated from active pol I and is thought to be essential for utilization of the murine ribosomal gene promoter. Genes encoding this factor have not yet been identified. Other pol I-specific transcription initiation factors have been described to be involved in the regulation of rRNA synthesis of higher eukaryotes. The upstream binding factor (UBF), was demonstrated to be a target molecule involved in the up- and downregulation of rRNA synthesis. The ratio of phosphorylated to non-phosphorylated UBF was suggested to define its transactivation properties (O'Mahony et al., 1992a,b; Voit et al., 1992). Furthermore, the retinoblastoma susceptibility gene product was shown to interact with UBF, resulting in the repression of pol I-dependent transcription (Cavanaugh et al., 1995; Voit et al., 1997). Recently, SV40 large T antigen, a viral protein which can stimulate cell proliferation, was shown to activate pol I-dependent transcription by its interaction with the basal pol I-specific transcription factor SL1 (Zhai et al., 1997). SL1 (human) (Comai et al., 1992, 1994) or TIFIB (mouse or Acanthamoeba) (Eberhard et al., 1993; Radebaugh et al., 1994) can direct multiple rounds of pol I recruitment to the promoter (Schnapp and Grummt, 1991; Goodrich and Tjian, 1994; Beckmann et al., 1995; Hempel et al., 1996). SL1 and TIFIB are members of a family of multisubunit general transcription factors [including TFIID (Dynlacht et al., 1991; Tanese et al., 1991; Zhou et al., 1992) and TFIIIB (Lobo et al., 1992; Taggart et al., 1992) containing the TATA-binding protein (TBP) in a stable complex with tightly associated proteins called TAFs. Finally, another pol I-specific transcription factor described in mouse, TIFIC, binds directly to polymerase and appears to mediate both initiation and transcript elongation (Schnapp et al., 1994). Its role in growth-dependent regulation of transcription remains to be established. Yeast pol I evidently requires two multisubunit complexes, namely CF and UAF, as well as a single-subunit transcription factor, Rrn3p, for maximal utilization of the ribosomal gene promoter in vitro (Keys et al., 1994, 1996; Yamamoto et al., 1996). Three essential genes (RRN6, RRN7 and RRN11) encode the polypeptide subunits of CF, each of which is required for specific initiation in vitro (Keys et al., 1994). UAF, which is stimulatory in vitro, contains Rrn5p, Rrn9p, Rrn10p and the histones H3 and H4 (Keener et al., 1997). UAF is thought to bind upstream of the ribosomal promoter (at the upstream element) to form a stable complex which apparently helps recruit CF to the core promoter element (Keys et al., 1996). CF may function analogously to mammalian SL1/TIFIB, which contains TBP (Keys et al., 1996). Both CF and UAF interact specifically with TBP (Lin et al., 1996; Steffan et al., 1996). On a template lacking a UAF-binding site, TBP is required for stimulation of transcription mediated by UAF but not for basal transcription (Steffan et al., 1996). Rrn3p, which is also required for in vitro transcription, was suggested to interact directly with pol I since pre-incubation of Rrn3p with pol I led to a stimulation of transcription (Yamamoto et al., 1996). Although all of the identified factors presumably cooperate with pol I at some point, little is known about how the polymerase itself interacts with the initiation factors. Recently, we described the resolution and characterization of distinct pol I populations from yeast whole-cell extracts using a reconstituted in vitro transcription system (Tschochner, 1996; Milkereit et al., 1997). Only a minor monomeric form of pol I was found to be active in promoter-driven transcription, whereas the bulk of pol I existed as inactive monomers or dimers. Here we show that the initiation-competent pol I population (<2% of total pol I) can be purified further as a complex in stable association with the essential initiation factor Rrn3p. We provide evidence that formation of the pol I–Rrn3p complex and its dissociation during transcription can serve as a molecular switch for transcription initiation and growth rate-dependent regulation of rRNA synthesis. Results Initiation-competent monomeric pol I is stably associated with Rrn3p Our aim was to isolate yeast RNA polymerase I in an active form for promoter-dependent transcription. For the fractionation scheme diagrammed in Figure 1, pol I purification was monitored with three different assays: (i) quantitative immunoblotting with antibodies directed against the pol I-specific subunit A49 or A190 (kindly provided by A.Sentenac and colleagues); (ii) non-specific RNA chain elongation with single-stranded or nicked DNA templates (Roeder, 1974); and (iii) a promoter-dependent run-off transcription assay performed in the presence of two other essential fractions. One of the two accessory fractions (designated TBP-cpl in Figure 1) contains a 240 kDa protein complex that includes TBP; the other (fraction B600) is a crude fraction that lacks pol I activity. As shown previously, the polymerase-containing fraction (B2000; Figure 1) resolved into three fractions by size-exclusion chromatography. Two of these were inactive for promoter-dependent transcription: one contained predominantly dimers while the other contained the majority of monomeric pol I. A third fraction contained monomeric pol I that was active for promoter-dependent transcription (Milkereit et al., 1997). On this and other sizing columns, initiation-competent pol I migrated with a slightly higher molecular mass than did the bulk of (inactive) monomeric pol I, providing the first indication that additional subunit(s) might be associated with the active form of the enzyme (Milkereit et al., 1997). Figure 1.Purification scheme of pol I and fractions used for the reconstituted in vitro transcription from yeast whole-cell extracts. Download figure Download PowerPoint Rrn3p is known to be essential for promoter-directed yeast pol I transcription (Yamamoto et al., 1996). To determine the fate of Rrn3p in our fractionation scheme, fractions were analysed by immunoblotting with affinity-purified antibodies specific for Rrn3p. Both Rrn3p and pol I were detected in the B2000 fraction (data not shown). Proteins in this fraction were resolved further on a Superose-6 gel filtration column in the presence of 1.5 M potassium acetate. Even under these stringent conditions, Rrn3p and monomeric pol I co-eluted from the column (Figure 2A, upper and lower panels). No Rrn3p was detected in fractions eluting at the volume expected for monomeric Rrn3p (with a predicted molecular mass of 72 kDa; data not shown). We performed two additional experiments to confirm that promoter-dependent initiation activity co-purified with Rrn3p in association with pol I. First, pol I, Rrn3p and initiation activity were co-purified by immunoaffinity chromatography exploiting a haemagglutinin (HA)-tagged AC40 pol I subunit (Figure 2B). In addition, pol I and Rrn3p also were co-purified by metal chelate affinity-chromatography using a histidine-tagged ABC23 pol I subunit (data not shown). Taken together, these data strongly suggest that Rrn3p is a component of a stable pol I enzyme complex that supports accurate transcription initiation in vitro. Figure 2.Rrn3p is stably associated with monomeric initiation-competent pol I. (A) Gel filtration of initiation-competent pol I (50 μl of fraction B2000) with a Superose-6 column in the presence of 1.5 M potassium acetate. A 1.5 μl aliquot of the 240 kDa, TBP-containing protein complex and 1 μl of fraction B600 were added to 1.5 μl of each fraction from the column for the reconstituted transcription assay (middle panel). Each 40 μl and 4 μl were tested for Western blot analysis (lower panel) and non-specific RNA synthesis (upper panel), respectively. (B) Co-immunopurification of Rrn3p with pol I. Fraction B2000 was incubated with HA-specific antibodies attached to Sepharose beads (BAbCO). After washing with buffers containing 600 mM potassium acetate (see Materials and methods) (lanes 2 and 3), the column was eluted with 1 mg/ml HA-peptide dissolved in the same acetate buffer (lanes 4 and 5). Then 1% of the load and 10% of each fraction were separated on 10% SDS–PAGE and silver stained (upper panel) or blotted onto a PVDF membrane (Millipore) and developed with anti-Rrn3p antibodies (lower panel). Specific transcriptional activity of 1% of the load and 5% of each fraction is depicted in the middle panel. Download figure Download PowerPoint Only a minor proportion of yeast RNA polymerase I is competent for initiation Initiation-competent pol I from the sizing column (Milkereit et al., 1997) was applied to a MonoQ column and eluted with a salt gradient (Figure 3A). More than 75% of the specific transcription activity loaded onto the column was recovered. The peak fractions of pol I protein, as determined by immunoblotting (fractions 20 and 21), did not coincide with promoter-dependent transcription activity. The peak of specific activity eluted in fraction 22 (Figure 3A, lower panel), which contained <15% of the polymerase, as determined by Western blot analysis. Evidently, monomeric pol I was resolved into two populations on the MonoQ column, only one of which was active in promoter-driven transcription. We tested this fraction for Rrn3p content; as in the gel filtration experiments, initiation activity coincided with the appearance of Rrn3p (Figure 3A, middle and lower panels). Titrating initiation-incompetent pol I (MonoQ fraction 20) into Rrn3p-containing fractions (e.g. MonoQ fraction 23) neither stimulated nor inhibited specific initiation (Figure 3A, lane 8). This result showed that the weak promoter-dependent activity of fractions containing the highest concentrations of pol I (fractions 20 and 21) was not due to the presence of an inhibitor, and that the total amount of pol I was not limiting in the strongly active Rrn3p-containing fractions. SDS–PAGE analysis of MonoQ fractions (Figure 3B) showed comparable degrees of purity in peak fractions for non-specific (Figure 3B, lane 1) and promoter-specific pol I activity (Figure 3B, lane 2). However, in addition to the typical pattern of pol I subunits, a few other polypeptide bands were unique to fraction 22 (Figure 3B). One of these corresponded to a polypeptide with an apparent molecular mass of 72 kDa, consistent with the predicted mass of Rrn3p (Figure 3B, lane 2), which was recognized by the anti-Rrn3p antibodies. We conclude that only a small proportion of yeast pol I can be isolated from whole-cell extract in an initiation-active form, and that this initiation-competent enzyme fraction contains Rrn3p. Figure 3.A small proportion of pol I is associated with Rrn3p. Co-purification of Rrn3p and transcriptional activity on Mono Q. Monomeric pol I which had been separated from pol I dimers on a Sephacryl S-300 column was loaded onto a MonoQ column and eluted at ∼1.1 M potassium acetate applying a linear gradient from 600 to 1300 mM potassium acetate. (A) Two μl of each fraction were tested in non-specific RNA synthesis (upper panel) and promoter-dependent transcription (lower panel) in the presence of 1.5 μl of TBP-cpl and 1 μl of fraction B600, respectively. In the assay illustrated in lane 8, 1 μl of fraction 20 was mixed with 1 μl of fraction 23 before starting the transcription reaction. Five μl of each fraction were analysed by Western blotting with antibodies against the pol I-specific subunit A49 and against Rrn3p (middle panels). (B) Eighty percent each of the peak fractions from a MonoQ column in non-specific activity of RNA synthesis (lane 1) and promoter-dependent activity (lane 2) were separated on an 8% SDS–polyacrylamide gel and silver stained. The positions of pol I subunits and the size of Rrn3p (72 kDa) are indicated. Download figure Download PowerPoint A pol I–Rrn3p complex represents a subform of pol I highly active in initiation Immunoaffinity purification of the pol I–Rrn3p complex from the B2000 fraction using antibodies directed against the N-terminal peptide of Rrn3p allowed a more detailed analysis of initiation-competent pol I. After elution of the co-immunoprecipitated complexes with an excess of the N-terminal Rrn3p peptide, SDS–PAGE analysis revealed a seemingly stoichiometric relationship between pol I subunits and a polypeptide of the apparent molecular mass of 72 kDa, which obviously resembled Rrn3p (Figure 4A; the stoichiometry is inferred from silver staining intensity, which may not reflect accurately the relative abundance of these particular proteins). No other proteins in the 65–200 kDa mass range were visible, indicating that additional polypeptides in this range present in the initiation-active MonoQ fraction 22 (Figure 3B) were not required for promoter-dependent initiation. More importantly, the immunopurified pol I–Rrn3p complex possessed a very high specific activity, with 3–4 ng of the purified complex being sufficient to saturate the reconstituted transcription assay (Figure 4A, lower panel). This corresponded to a specific activity of 15 pmol transcripts per mg of pol I. Good recovery of specific activity through all purification steps indicated that the putative pol I–Rrn3p complex identified by these experiments is highly stable (Table I). The large increase in specific activity of the pol I–Rrn3p complex during purification evidently was not due to the loss of inhibitory activities during the purification procedure. Mixing crude pol I-containing fractions (K350, T0, B2000) with initiation-competent pol I did not reduce the yield of transcripts in the reconstituted assay (data not shown). Quantitative immunoblotting and transcription assays revealed that <2% of the pol I present in the B2000 fraction resided in the pol I–Rrn3p complex. Figure 4.Immunoprecipitated Rrn3p is highly active in transcription initiation if it is incorporated in a pol I–Rrn3p complex. (A) Immunoprecipitation was performed with anti-Rrn3p antibodies as described in Materials and methods. After elution of the precipitated complex with an excess of the N-terminal Rrn3p peptide, half of the last wash step (lane 3) and half of the eluate (lane 4) were separated on a 10% SDS–polyacrylamide gel and stained with silver (upper panel). The purified pol I-A (200 ng) kindly provided by A.Sentenac and colleagues is depicted in lane 1. The sizes of the two largest pol I subunits and of Rrn3p are indicated. Lower panel: transcription initiation assay with the eluted pol I–Rrn3p complex. One μl of fraction B600 was added to 1 μl of fraction B2000 (containing 200 ng of pol I) or to 2 and 4 μl of the eluate (containing 2 and 4 ng of pol I, respectively) in either the presence (lane 6) or absence (lanes 4 and 5) of 1.5 μl of TBP-cpl and assayed for transcription initiation. Control reactions with the same amounts of fractions B600 and/or TBP-cpl and 4 μl of the wash step are shown in lanes 2 and 3. Note the reduction of non-specific radioactivity in lane 6 in comparison with lane 1. (B) Eighty μl of fraction B2000 (lanes 1 and 3) and 200 μl of fraction T0 were incubated with 40 μl of protein A–Sepharose beads covered with anti-Rrn3p antibodies (lanes 1 and 2) or empty beads (lanes 3). Half of each washed complex was subjected to Western blot analysis (left) or to transcription reactions in the presence of 1 μl of fraction B600 and 1.5 μl TBP-cpl (right). Western blots were developed with anti-pol I antibodies (directed against subunit A190) (upper panel) and anti-Rrn3p antibodies (lower panel). Download figure Download PowerPoint Table 1. Purification of initiation-competent pol I from yeast whole-cell extracts Fraction/step Protein (mg) Volume (ml) Activity (U) Specific activity (U/mg) Relative specific activity Whole-cell extract 2100 450 K350 900 200 12 800 14.2 1 PA600 60 5 17 500 292 21 B2000 8.25 2.3 9800 1190 84 Sephacryl S-300 0.6 6 2570 4283 302 MonoQ PC 1.6/5 0.042 0.5 1950 46 428 3270 One unit is defined as the amount required to produce 0.1 fmol of accurately initiated transcripts in the reconstituted initiation assay. Initiation activity could not be detected in whole-cell extracts and was apparently diminished in fraction K350 due to the presence of inhibitors. The protein concentration after the MonoQ column was estimated by silver-stained SDS gels and by Western blotting. Nomura and colleagues reported that the majority of Rrn3p in crude extracts is monomeric (Yamamoto et al., 1996). Immunoprecipitation with antibodies directed against the N-terminus of Rrn3p confirmed that the majority of Rrn3p is not associated with the initiation-competent pol I complex: a large proportion of Rrn3p was immunoprecipitated from fractions that were inactive in pol I-dependent transcription (such as T0) with no co-precipitation of pol I (Figure 4B, left panel, lane 2). However, when Rrn3p was immunoprecipitated from fractions containing initiation-competent pol I, such as K350 and PA600 (data not shown) or B2000 (Figure 4B, left panel, lane 1), a significant proportion of pol I was co-precipitated. After extensive washing, immunoprecipitated pol I–Rrn3p complexes were assayed for promoter-dependent transcription by adding template, fractions B600, the TBP complex and nucleotide substrates to the beads (Figure 4B, right panel, lane 1). Although a similar amount of Rrn3p was precipitated from fractions B2000 and T0, efficient initiation of rRNA synthesis was restricted to immunoprecipitated pol I–Rrn3p complexes from fraction B2000. The slightly elevated transcriptional activity visible in Figure 4B, lane 2 (right panel) is probably due to some pol I–Rrn3p complexes still present in fraction T0. Indeed, long exposures of the Western blot depicted in Figure 4B (left panel) also showed trace amounts of co-precipitated pol I in lane 2. No initiation activity co-precipitated with empty beads (Figure 4B, lane 3). Consistent with published results (Yamamoto et al., 1996), initiation activity could be detected when immunoprecipitated Rrn3p from the transcriptionally inactive fraction T0 (which was not complexed with pol I) was supplemented with ∼3 μg of initiation-inactive pol I, which did not contain Rrn3p (fraction 20 of the MonoQ column) (data not shown). However, transcription efficiency was insignificant compared with that of the pol I–Rrn3p complex isolated from fraction B2000, even with a 1000-fold greater amount of pol I and a large excess of Rrn3p. The observation that the majority of Rrn3p is not associated within the initiation-competent pol I complex suggests that either pol I or Rrn3p has to be modified to enable an interaction between the two partners. Indeed, analysis of Rrn3p-containing yeast fractions on two-dimensional gels revealed more than two different charged populations of Rrn3p (data not shown). However, a possible correlation with their activities could not been deduced thus far, since the pol I-associated Rrn3p failed to migrate into the first dimension of the gel. Taken together, these data strongly suggest that a distinct pol I complex, consisting of pol I core enzyme, Rrn3p and possibly another associated factor(s), is formed either prior to or simultaneously with the start of promoter-dependent rRNA synthesis. Genetic and biochemical analyses have shown that Rrn3p activity is necessary for transcription initiation (Yamamoto et al., 1996). We propose that additional factors and/or modifications of Rrn3p or pol I are required to form a functional pol I–Rrn3p complex. While the conditions required for formation of the pol I–Rrn3p complex remain unknown, our results clearly show that only a pre-formed complex is able to initiate transcription efficiently in vitro. During transcription the pol I–Rrn3p complex is disrupted and its capacity to initiate rRNA synthesis is exhausted Although the pol I–Rrn3p complex was stable during purification and extended incubation in buffers used for in vitro transcription, the complex appeared to disintegrate during transcription. pol I could be still co-immunoprecipitated with Rrn3p after a 1 h incubation with all transcription components except the template (Figure 5A, lane 2) or nucleotide substrates (Figure 5A, lane 3). In contrast, when transcription was allowed to proceed for 1 h, pol I no longer co-precipitated with Rrn3p (Figure 5A, lane 4). Gel filtration experiments previously had demonstrated that no free Rrn3p was present in the fractions used for the reconstituted assay before in vitro transcription (data not shown), indicating that all Rrn3p that could be immunoprecipitated after the transcription reaction without pol I was indeed released from the pol I–Rrn3p complex. Figure 5.Dissociation of pol I–Rrn3p complex during one round of transcription and loss of its transcriptional activity. (A) Co-immunoprecipitation of Rrn3p and pol I before and after transcription. Transcription assays containing 40-fold amounts of all ingredients (40 μl of fraction B600, 60 μg of fraction B2000) were performed in the absence of template (lane 2) or nucleotides (lane 3), or in the presence of 4 μg of template and 0.2 mM NTPs (lane 4). After 60 min incubation at 25°C, immunoprecipitation was carried out with anti-Rrn3p antibodies. Precipitated proteins were separated on a 10% SDS–polyacrylamide gel, blotted onto PVDF membranes and screened with antibodies directed against the largest subunit of pol I, A190 (upper panel), and Rrn3p (lower panel). Five μg of fraction B2000 was blotted on lane 1. (B) The pol I–Rrn3p complex loses its ability to start rRNA synthesis during ongoing transcription. As indicated in the scheme at the bottom, template E (pSES5 linearized with EcoRV) was pre-incubated with fraction B600, TBP-cpl and the pol I–Rrn3p complex (pol I-i) (MonoQ fraction 22) and transcription was started with the addition of nucleotides. At the indicated time points (lanes 1–6), template B (pSES5 linearized with BamHI) was added together with fraction B600, TBP-cpl and fresh NTPs and incubated for a further 30 min. In lane 6, pol I-i was added together with the second template, fraction B600 and TBP-cpl after transcription of template E had proceeded for 60 min. Lanes 7 and 8 show control reactions lacking either template E (lane 7) or nucleotides (lane 8) during the first 60 min of incubation. (C) In vitro generated run-off transcripts are synthesized in one single round of transcription. Transcription was performed on immobilized templates which contained the pol I promoter, but lacked cytidine within the first 34 nucleotides form the start site. Lanes 1–3: in vitro reconstituted transcription in the absence (ctrl, control, lane 1) or in the presence of 0.5 mg/ml heparin or 0.025% Sarkosyl (lanes 1 and 2, respectively). Heparin and Sarkosyl were added to the template prior to the protein fractions. Lanes 4–12: a pre-initiation complex was formed with fractions B600, pol I-i and TBP-cpl on the immobilized template for 20 min. The pre-initiation complexes were washed to remove unbound pol I and transcription factors. ATP, [32P]GTP and UTP were added to generate a ternary elongation complex (Tschochner and Milkereit, 1997). After a further 20 min of incubation, CTP and either heparin or Sarkosyl were added to give final concentrations of 0.5 mg/ml and 0.025%, respectively (lanes 4–7). Control reactions were performed without heparin or Sarkosyl (lanes 8–12). Transcription elongation was stopped after the indicated times. Download figure Download PowerPoint If co-immunoprecipitation experiments before and after transcription were performed using the HA-tagged pol I, an analogous result was obtained (data not shown): co-immunoprecipitation of pol I and Rrn3p was observed exclusively before, but never after the transcription reaction. The effect of transcription on the stability of the pol I–Rrn3p complex was tested in an order-of-addition experiment (Figure 5B). After pre-incubation of template E with pol I–Rrn3p complex and all necessary transcription factors, transcription was started by the addition of nucleotide substrates; a second template (template B) that had been pre-incubated with fractions B600 and TBP-cpl was then added along with fresh nucleotides at various time points. When transcription of template E was allowed to proceed for >40 min (Figure 5B, lane 3–5), no transcripts were generated from the second template (template B), indicating depletion or sequestration of pol I–Rrn3p activity. 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• W2088808837 date "1998-07-01" @default.
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• W2088808837 title "A specialized form of RNA polymerase I, essential for initiation and growth-dependent regulation of rRNA synthesis, is disrupted during transcription" @default.
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