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• W2101005129 abstract "Article15 December 2005free access A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation Emilie Landrieux Emilie LandrieuxPresent address: CMU/Département de Microbiologie et de Médecine Moléculaire, rue Michel Servet 1, 11211 Genève, Switzerland Search for more papers by this author Nazif Alic Nazif Alic Search for more papers by this author Cécile Ducrot Cécile Ducrot Search for more papers by this author Joël Acker Joël Acker Search for more papers by this author Michel Riva Corresponding Author Michel Riva CEA/Saclay, Laboratoire de Transcription des Gènes, Service de Biochimie et de Génétique Moléculaire, Gif sur Yvette, France Search for more papers by this author Christophe Carles Christophe Carles Search for more papers by this author Emilie Landrieux Emilie LandrieuxPresent address: CMU/Département de Microbiologie et de Médecine Moléculaire, rue Michel Servet 1, 11211 Genève, Switzerland Search for more papers by this author Nazif Alic Nazif Alic Search for more papers by this author Cécile Ducrot Cécile Ducrot Search for more papers by this author Joël Acker Joël Acker Search for more papers by this author Michel Riva Corresponding Author Michel Riva CEA/Saclay, Laboratoire de Transcription des Gènes, Service de Biochimie et de Génétique Moléculaire, Gif sur Yvette, France Search for more papers by this author Christophe Carles Christophe Carles Search for more papers by this author Author Information Emilie Landrieux‡, Nazif Alic‡, Cécile Ducrot, Joël Acker, Michel Riva 1 and Christophe Carles 1CEA/Saclay, Laboratoire de Transcription des Gènes, Service de Biochimie et de Génétique Moléculaire, Gif sur Yvette, France ‡These authors contributed equally to this work *Corresponding author. CEA/Saclay, Laboratoire de Transcription des Gènes, Service de Biochimie et de Génétique Moléculaire, F-91191 Gif sur Yvette Cedex, France. Tel.: +33 1 69 08 84 17; Fax: +33 1 69 08 47 12; E-mail: [email protected] The EMBO Journal (2006)25:118-128https://doi.org/10.1038/sj.emboj.7600915 Present address: CMU/Département de Microbiologie et de Médecine Moléculaire, rue Michel Servet 1, 11211 Genève, Switzerland PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info While initiation of transcription by RNA polymerase III (Pol III) has been thoroughly investigated, molecular mechanisms driving transcription termination remain poorly understood. Here we describe how the characterization of the in vitro transcriptional properties of a Pol III variant (Pol IIIΔ), lacking the C11, C37, and C53 subunits, revealed crucial information about the mechanisms of Pol III termination and reinitiation. The specific requirement for the C37–C53 complex in terminator recognition was determined. This complex was demonstrated to slow down elongation by the enzyme, adding to the evidence implicating the elongation rate as a critical determinant of correct terminator recognition. In addition, the presence of the C37–C53 complex required the simultaneous addition of C11 to Pol IIIΔ for the enzyme to reinitiate after the first round of transcription, thus uncovering a role for polymerase subunits in the facilitated recycling process. Interestingly, we demonstrated that the role of C11 in recycling was independent of its role in RNA cleavage. The data presented allowed us to propose a model of Pol III termination and its links to reinitiation. Introduction Under conditions of multiple round transcription by DNA-dependent RNA polymerases, initiation, elongation, and termination are the three elementary steps of the first transcription cycle, whereas reinitiation, elongation, and termination are related to the subsequent cycles. Transcription termination implies the proper recognition of the terminator elements and the release of the newly synthesized transcript. Our knowledge of the molecular mechanisms driving termination depends on the system studied. The three transcription machineries seem to have evolved different strategies for transcription termination. Eukaryotic mRNAs possess poly(A) signals that define their 3′ ends and mediate downstream transcription termination by Pol II (Proudfoot, 1989). The tight coupling of termination to mRNA 3′-end processing and polyadenylation involves the regulated interaction between 3′ end processing activities and the carboxy-terminal domain of the largest subunit of Pol II (McCracken et al, 1997; Dichtl et al, 2002; Proudfoot et al, 2002; Zhang et al, 2005). In addition, chromatin remodeling of termination regions might be required for Pol II transcription termination (Alen et al, 2002). While the complete set of molecular mechanisms driving Pol II transcription termination remains to be fully elucidated, the requirement for the pausing of the enzyme over the terminator region to ensure termination of Pol II transcription is firmly established (Birse et al, 1997; Dye and Proudfoot, 2001). The mechanism of Pol I termination is different and involves cis- and trans-acting factors required to properly terminate and to release the transcript. Termination by Pol I minimally requires a sequence-specific DNA-binding protein that acts as a roadblock to pause the elongating polymerase, and a release element upstream of the pause site (Bartsch et al, 1988; Lang et al, 1994; Evers and Grummt, 1995; Zhao et al, 1997). In all cases, termination occurs some 10–12 bp upstream of the orientation-sensitive DNA-binding site of the termination factor. In mouse and yeast, the upstream element works in concert with a cellular factor or release factor that interacts with Pol I and drives the dissociation of the paused ternary complexes (Tschochner and Milkereit, 1997; Jansa et al, 1998; Jansa and Grummt, 1999). At variance with Pol I and II, Pol III accurately and efficiently recognizes termination signals constituted by a short stretch of thymidine residues in the apparent absence of any additional components (Cozzarelli et al, 1983). The observation that Pol III pauses at uridine stretches, and in particular at the terminator (Campbell and Setzer, 1992; Matsuzaki et al, 1994), strongly suggests that pausing of the enzyme is a general prerequisite for transcription by the three forms of the eukaryotic RNA polymerase. Consequently, it has been proposed that the intrinsic RNA cleavage activity of Pol III, mediated by the C11 subunit, plays a role in the termination process by raising the dwell-time of the enzyme on the terminator, and thereby favoring the release of the RNA and the dissociation of the complex (Chédin et al, 1998). An important feature of yeast class III gene transcription is the direct coupling between termination and reinitiation of transcription. This process, called facilitated reinitiation, is characterized by the commitment of Pol III to reinitiate more rapidly on the same gene after the first transcription cycle without being released and results in a higher initiation efficiency (Dieci and Sentenac, 1996). Facilitated recycling pathway requires the termination to take place at the natural termination signal and probably involves protein–protein interactions between Pol III and components of the preinitiation complex (Ferrari et al, 2004). Despite recent advances in our understanding of facilitated reinitiation, whether a particular polymerase subunit(s) is involved in this mechanism remains unknown. In this study, we characterize an incomplete form of Pol III (Pol IIIΔ) lacking the C11, C37, and C53 subunits, and which displays strong transcriptional defects. Add-back experiments with combinations of the three subunits to Pol IIIΔ highlighted the key role of the C37–C53 complex in the recognition of the terminator elements, and demonstrated that the RNA cleavage activity of Pol III, mediated by the C11 subunit, was not involved in this step, nor in the release of the newly synthesized transcript. In contrast, the C11 subunit was required for the enzyme to perform multiple rounds of transcription. In addition, the RNA-cleavage activity of Pol III was not required for reinitiation, demonstrating a dual function for the C11 subunit. The finding that Pol III transcription termination and reinitiation require distinct, but interacting subunits, demonstrates that these two steps of the transcriptional cycle can be uncoupled. The data obtained are discussed in a model of Pol III transcription termination and reinitiation. Results Among the 17 subunits of the yeast RNA polymerase III (Pol III), RPC37, the gene encoding the C37 subunit was the last to be characterized. Peptidic sequence of internal tryptic fragments of the 37 kDa component of the yeast Pol III allowed us to identify the YKR025w open reading frame (ORF). The same ORF (renamed RPC37) was independently isolated in a two-hybrid screen with the Pol III subunit C53 as a bait (Flores et al, 1999). The interaction between the 37 kDa polypeptide and C53 was further demonstrated by pull-down and co-immunoprecipitation experiments (data not shown). The status of this polypeptide (named hereafter C37) as a genuine Pol III subunit was finally confirmed by the presence of its orthologue HsRPC5 in the human Pol III (Hu et al, 2002). The interaction between HsRPC5 and HsRPC53, the human orthologue of C53, is also conserved, paralleling the association of yeast C37 and C53 subunits, and is mediated by the domains conserved within their yeast counterparts (Hu et al, 2002). C37 was shown to be essential for yeast cell viability, similarly to all components of the class III transcription machinery. The C37 subunit has no paralogue in the two other nuclear RNA polymerases, and is therefore one of the five subunits specific to Pol III. Mutational analysis of RPC37 A detailed analysis of the C37 amino-acid sequence did not reveal any conserved protein motif that might point out a particular function for the protein within the transcriptional process. Therefore, to functionally characterize this polypeptide we generated mutants by random mutagenesis of RPC37. Among four rpc37 alleles isolated that conferred a thermosensitive growth phenotype, we focused our interest on the most thermosensitive rpc37-1 allele. This allele was characterized by a semiconservative mutation resulting in the I141V change, and a second point mutation introducing a premature stop codon that led to the expression of a polypeptide lacking the 27 C-terminal residues. Cells expressing this mutant version of C37 were unable to grow at 34°C (Figure 1A). To facilitate the biochemical analysis of Pol III containing the mutated subunit and to overcome the lack of specific antibodies raised against the C37 subunit (Huet et al, 1985), we constructed a mutant strain (C37HAΔCt) which contained the rpc37-1 allele corrected for the semiconservative point mutation at position 141 and fused to a sequence encoding three HA epitopes at its 3′ end. A control strain (C37HA) expressing the corresponding C-terminally HA tagged form of the wild-type C37 subunit was also constructed. As shown in Figure 1A, the C37HAΔCt strain was not able to grow at 34°C, similarly to the rpc37-1 containing strain, whereas the control C37HA did not display any growth defects, indicating that the presence of the C-terminal tag did not affect growth. Collectively, these data show that the C-terminal truncation of the C37 subunit is alone responsible for the thermosensitive phenotype observed. Figure 1.Pol IIIΔ lacks the C11, C37, and C53 subunits. (A) Growth of the control wild-type strain (C37HA), and of the C37HAΔCt and the rpc37-1 mutant strains at 30 and 34°C. C37 subunit expressed in each strain is illustrated schematically on the right. Dark gray—HA tag, *—point mutation. (B) Pol III purified from the control wild-type strain expressing an HA-tagged version of C37 (lane 1), from the C37HAΔCt mutant strain (lane 2), and from the SpC11-C37HA mutant strain (lane 3) were analyzed by electrophoresis on a 13% SDS–polyacrylamide gel and silver-staining. Subunits are indicated on the left, *—major contaminants. (C) Western blot analysis of Pol III (lanes 1, 2, and 3 as in B) with a mixture of antibodies directed against the HA epitope and the Pol III subunits C34 or C53. Anti-C34 antibodies are used to control even Pol III loading. (D) Rescue of the thermosensitivity of the mutant rpc37-1 strain with plasmids overexpressing C11, C37, or C53. Corresponding empty vectors are shown as controls. Spots are formed from 10 μl of cells incubated for 2 days. Download figure Download PowerPoint Identification of an incomplete form of Pol III lacking the C11, C37, and C53 subunits To characterize the in vitro transcriptional properties of Pol III containing the truncated version of the C37 subunit, the enzyme was purified from the C37HAΔCt strain and from a control strain according to the usual micropurification procedure (Huet et al, 1996). Subunit composition of the wild-type and mutant enzymes was analyzed by SDS–PAGE followed by silver staining. The C11 subunit was not detected in the mutant Pol III (Figure 1B, lane 2). Since no antibodies against this polypeptide are available, the absence of C11 was confirmed by analyzing the RNA cleavage activity of the enzyme, which strictly depends on the presence of the C11 subunit (Chédin et al, 1998). Mutant Pol III purified from the C37HAΔCt strain did not exhibit any RNA cleavage activity, confirming that the C11 subunit was indeed missing (data not shown). In addition, and despite the presence of a number of contaminants at the C37 and C53 subunit level, these two subunits seemed to be absent, or at least under-represented (Figure 1B, lanes 1 and 2). To confirm these observations, Western blot analysis was performed with anti-HA antibodies (detecting the HA-tagged C37 subunit) and anti-C53 antibodies. Whereas C37 and C53 subunits were present in the control enzyme preparation (Figure 1C, lane 1), they could not be detected in the mutant Pol III (Figure 1C, lane 2). These results demonstrated that Pol III purified from the mutant C37HAΔCt strain was deprived of the three subunits C11, C37, and C53, and suggested that C37 played a role in the association of C11 and C53 within the enzyme. The effect of the RPC37 mutation on the stability of the C53 subunit within the Pol III complex is likely explained by the direct interaction between the C37 and C53 polypeptides (see above and Flores et al, 1999). The destabilization of the association of C11 subunit was more surprising because no interaction could be demonstrated by two-hybrid and Far-Western between C11 and C37 (data not shown), nor between C11 and C53 (Chédin et al, 1998; Flores et al, 1999). To confirm the existence of a link between the C11, C37, and C53 subunits by a genetic approach, we tested whether we could suppress the thermosensitive phenotype conferred by different rpc37 alleles upon overexpression of C11 and/or C53. As shown in Figure 1D, the overexpression of the C11 subunit allowed growth of the rpc37-1 strain at 34°C. A similar result was obtained by the overexpression of the C53 subunit (Figure 1D). This suppression was allele specific (data not shown). These genetic interactions confirmed the link between C37, C11, and C53 subunits and suggested that the thermosensitive phenotypes of the mutant rpc37 strains resulted from a defect in the assembly and/or stability of the complete form of Pol III in vivo. Interestingly, these results were reminiscent of the characterization of an incomplete form of Pol III (named Pol IIIΔ) lacking the C11 subunit and containing a substoichiometric amount of the C53 subunit (from 0 to 20% depending on the preparation), obtained upon purification of the enzyme from a yeast strain in which a deletion of RPC11 was complemented by its Schizosaccharomyces pombe orthologue (Chédin et al, 1998). Whether the C37 subunit was present or not in Pol IIIΔ could not be determined at that time due to the absence of specific antibodies directed against C37 and to the comigration of the C37 and AC40 subunits during electrophoresis. To address this issue, we generated a yeast strain (SpC11-C37HA) carrying an HA-tagged RPC37 gene and a deletion of RPC11 heterocomplemented by the S. pombe C11 orthologue (see Materials and methods). Western blot analysis with anti-HA antibodies and silver staining clearly showed that Pol III purified from this strain lacked the C37 subunit in addition to C11 and C53 (Figure 1B, lane 3, and Figure 1C, lane 3). Thus, the same incomplete form of Pol III is obtained from either a strain mutated in RPC11 or in RPC37. From these results, we propose a new definition of Pol IIIΔ as an incomplete form of Pol III deprived of the three subunits C11, C37, and C53. Transcriptional properties of the Pol III lacking subunits C11, C37, and C53 Pol IIIΔ was active when assayed in an in vitro promoter-independent transcription assay with poly [d(A-T)] as template, indicating that the C11, C37, and C53 subunits were not required for the basal process of RNA synthesis (data not shown). As previously reported, Pol IIIΔ was found to have a number of enzymatic defects in vitro. It initiated correctly in specific transcription assays but was deficient in termination and totally lacked the RNA cleavage activity intrinsic to Pol III. We formerly proposed the existence of a direct link between the termination defect and the absence of intrinsic RNA cleavage activity of Pol IIIΔ, but in apparent disagreement with this hypothesis, incubation of the recombinant C11 subunit (rC11) with Pol IIIΔ had no influence on its termination defect, whereas the RNA cleavage activity of purified ternary complexes was fully restored (Chédin et al, 1998). To understand the molecular mechanism that controls transcription termination of class III genes, we performed add-back experiments with purified recombinant C11, C37, and C53 subunits using the SUP4 gene as a template in an in vitro specific-transcription assay. Note that all the results described below were identical whether the recombinant C37 and/or C53 subunits were purified from baculovirus-infected insect cells or from Escherichia coli cells (recombinant C11 was solely purified from bacterial cells). The terminator of the SUP4 gene is a T7GT6 sequence in the nontranscribed strand of the DNA (Figure 2A), and wild-type Pol III mostly terminated within the T7 stretch (Figure 2B, lane 1). As previously observed, a significant proportion of Pol IIIΔ molecules read-through the T7 stretch (hereafter called T1), and terminated within the T6 stretch (T2). The individual addition of any of the three subunits had no influence on this termination defect (Figure 2B). In sharp contrast, when increasing amounts of purified recombinant (C37–C53) complex were added back, the enzyme terminated within the T1 stretch only, as did the wild-type Pol III (Figure 2C). The same result was obtained when Pol IIIΔ was preincubated with a mixture of the two individual rC37 and rC53 subunits (data not shown). Note that while the r(C37–C53) complex triggered a correct recognition of the SUP4 gene terminator by Pol IIIΔ, the level of transcription was much lower than that observed for the wild-type enzyme (Figure 2C, compare lanes 1 and 5). Figure 2.The terminator-recognition defect of Pol IIIΔ is corrected by addition of the r(C37–C53) heterodimer but not by the individual C11, C37, or C53 subunits. (A) Schematic representation of the SUP4 terminator with the T1 and T2 blocks. (B) The products of in vitro transcription reactions carried out using the SUP4 template and either the Pol IIIwt (lane 1), the Pol IIIΔ (lane 2), or the Pol IIIΔ preincubated for 10 min with an excess of recombinant C37, C53, or C11 subunits (lanes 3, 4, and 5, respectively) were separated by electrophoresis and autoradiographed. (C) Reactions as in (B) were performed with Pol IIIwt (lane 1), Pol IIIΔ (lane 2), or Pol IIIΔ preincubated with increasing amounts of the r(C37–C53) complex (lanes 3–5). Position of full-length transcripts terminated at T1 or T2 is indicated. Download figure Download PowerPoint Overall, these data indicate that both C37 and C53 subunits play a critical role in the recognition of the terminator of class III genes by Pol III. Furthermore, they demonstrate that RNA cleavage is not required for correct recognition of the terminator elements by Pol III. Pol IIIΔ elongates more efficiently than wild-type enzyme The above observations prompted us to determine the mechanisms underlying the correction of Pol IIIΔ termination defect by the r(C37–C53) complex. We have already observed that the defect in terminator recognition by Pol IIIΔ could be corrected by decreasing the UTP concentration in the transcription reaction (Chédin et al, 1998). The rationale for this experiment was to decrease the elongation rate of the enzyme, in particular at the terminator. Since the r(C37–C53) complex restored accurate termination to Pol IIIΔ, we examined whether this complex had an effect on the elongation rate of the enzyme. Labeled ternary complexes containing wild type or Pol IIIΔ halted on the SUP4 gene at position +17 were generated (see Supplementary data), and purified by gel permeation to remove labeled nucleotides. Transcription elongation was next resumed upon addition of the four unlabeled nucleotides. Time course analyses of elongation of labeled 17-mer transcripts obtained with either wild-type Pol III or Pol IIIΔ in the absence or presence of the r(C37–C53) complex were performed, and labeled, full length, SUP4-o-tRNATyr transcripts were visualized by gel electrophoresis and autoradiography (Figure 3A). The amounts of newly synthesized full-length transcript were quantified, plotted as a function of time and fitted to a single exponential curve to determine the apparent rate constant (ka) for the elongation reaction (Figure 3B). Consistent with our hypothesis, Pol IIIΔ elongated faster than the wild-type enzyme (ka=0.13±0.02/s and ka=0.047±0.01/s, respectively). Remarkably, Pol IIIΔ incubated with the r(C37–C53) complex and the wild-type enzyme elongated with a similar apparent rate (ka=0.038±0.01/s and ka=0.047±0.01/s, respectively). These data indicate that the r(C37–C53) complex reduces the global elongation rate of Pol IIIΔ and concomitantly corrects the terminator-recognition defect of the enzyme. Figure 3.The r(C37–C53) heterodimer decreases the transcription elongation rate of Pol IIIΔ. (A) Time course analysis of elongation of radiolabeled 17-mer transcripts contained within the purified stalled ternary complexes of either Pol IIIwt or Pol IIIΔ to the full-length SUP4-o-tRNATyr. As indicated, Pol IIIΔ ternary complexes were either incubated or not with the r(C37–C53) complex for 10 min. Full-length SUP4-o-tRNATyr transcripts terminated at T1 or T2 are indicated, as well as those accumulated at the transitory pause sites (P1–P4). (B) Full-length transcripts were quantified and the values plotted as a function of time. Download figure Download PowerPoint The correct recognition of the terminator is not sufficient for transcription reinitiation The experiment described above showed that the addition of the r(C37–C53) complex to Pol IIIΔ did correct its terminator-recognition defect but resulted in a yield of SUP4-o-tRNATyr much lower than that obtained with the wild-type enzyme (Figure 2C, lanes 1 and 5). This observation was puzzling, because the experiment was performed under conditions where the enzyme was limiting, and where an equivalent number of transcriptionally competent Pol IIIΔ and wild-type Pol III molecules were used (data not shown). A simple explanation would be that, in the presence of r(C37–C53), Pol IIIΔ was either simply unable to reinitiate or was affected in facilitated reinitiation despite the correction of the terminator-recognition defect. To distinguish between these two possibilities, we used an equivalent number of transcriptionally competent Pol IIIwt and Pol IIIΔ molecules (Figure 4A) to estimate the amount of SUP4-o-tRNATyr synthesized by wild-type Pol III and Pol IIIΔ+r(C37–C53) during a 30-min time course, and we compared it to the amount of transcript synthesized by the wild-type enzyme under single-round conditions. Whereas the amount of transcript synthesized by the wild-type Pol III increased with time, the level of SUP4-o-tRNATyr synthesized by Pol IIIΔ in the presence of r(C37–C53) reached a plateau, after approximately 5 min of transcription, which corresponded to the level of single-round transcription by the wild-type enzyme (i.e. in the presence of heparin) (Figure 4B and C). Because in the presence of the C37 and C53 subunits, Pol IIIΔ elongates at the same rate as the wild-type enzyme (see Figure 3), these results demonstrate that Pol IIIΔ+r(C37–C53) performs only a single-round of transcription on the SUP4 gene under conditions where the wild-type enzyme carries out multiple rounds of transcription. This result suggests that the Pol IIIΔ+r(C37–C53) is affected either in transcription termination despite correct terminator recognition, or in transcription reinitiation (or in both). One difficulty is to determine, among the multiple molecular events taking place during these two consecutive steps, which of them belong to termination and which of them belong to reinitiation. For one particular transcription cycle, all molecular events taking place after the transcript release have no impact on that cycle but may affect the following cycle, and thus we consider them to belong to the reinitiation phase. Therefore, correct transcription termination only implies the proper recognition of the terminator elements and the normal release of the newly synthesized transcript. Since the above results demonstrated that Pol IIIΔ+r(C37–C53) properly recognized the terminator elements, we next investigated if it was affected in RNA release. Using immobilized SUP4 gene, we observed that the full-length transcript synthesized by the Pol IIIΔ in the presence of the C37 and C53 subunits was released in the supernatant with a timing undistinguishable from that observed when the wild-type enzyme transcribed this gene under single-round conditions (i.e. in the presence of heparin) (Figure 4D). Figure 4.Pol IIIΔ+r(C37–C53) properly terminates transcription but does not reinitiate. (A) The number of transcriptionally active ternary complexes formed by the quantities of Pol IIIwt (lane 1) and of Pol IIIΔ (lane 2) used in subsequent experiments was assessed by the formation of stalled complexes assembled on the SUP4 gene in the absence of GTP. Note that Pol IIIΔ incorporates an extra nucleotide at the 3′ end of the nascent RNA forming an 18-mer RNA, as previously reported (Chédin et al, 1998). (B) Time course analysis of SUP4 gene transcription performed with Pol IIIwt or with Pol IIIΔ+r(C37–C53). Single-round transcription was performed with Pol IIIwt in the presence of heparin for 5 or 30 min. (C) The amounts of full-length SUP4-o-tRNATyr transcripts in the time course analysis shown in (B) were quantified and the values plotted as a function of time. SR—single round. (D) Analysis of transcript release. Full-length transcripts contained in the supernatants of transcription reactions performed on an immobilized SUP4 template were separated by denaturing polyacrylamide gel electrophoresis, autoradiographed, and quantified. Transcription was carried out with either Pol IIIwt in the presence of heparin (single-round conditions) or Pol IIIΔ preincubated with r(C37–C53), and the reactions sampled at the times indicated. The values are given as a % of the final level of transcript released (which was similar in the two reactions examined) and plotted against time. Download figure Download PowerPoint We conclude that Pol IIIΔ+r(C37–C53) is not affected in transcription termination, and that the reason why this enzyme performs only single-round transcription is likely to be due to a transcription reinitiation defect. In the presence of C37 and C53, the C11 subunit, but not the RNA cleavage activity, is required for reinitiation by Pol III The above data demonstrated that the C11 subunit was not involved in terminator recognition, nor in the release of the newly synthesized transcript. However, the observation that an enzyme lacking only the C11 subunit (i.e. Pol IIIΔ+r(C37–C53)) was not able to reinitiate transcription brought to light the importance of C11 in the reinitiation process. When rC11 alone was added back to Pol IIIΔ, we did not observe a correction of the terminator recognition nor any effect on the reinitiation level (Figure 2B, lane 5). In sharp contrast, when rC11 was added to Pol IIIΔ+r(C37–C53), which alone completely failed to reinitiate transcription (see above), the amount of newly synthesized SUP4-o-tRNATyr transcript increased with time similarly to the wild-type enzyme (Figure 5A and B), demonstrating that Pol IIIΔ+rC11+r(C37–C53) could reinitiate transcription. To directly address whether the capacity to reinitiate conferred by rC11 onto Pol IIIΔ+r(C37–C53) resulted in facilitated reinitiation rather than simple reinitiation, a template competition assay was performed (Figure 6A). When a limiting amount of the enzyme was added to a mixture of GLU3 and SUP4 tRNA genes (each preassembled into a preinitiation complex) and multiple rounds of transcription allowed, Pol IIIΔ+rC11+r(C37–C53) equally transcribed the two templates (Figure 6B, lane 5), indicating that transcription of neither gene was intrinsically favored. In Figure 6B (lane 6), the enzyme was first sequestered in ternary complexes on the SUP4 template, and then multiple rounds" @default.
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• W2101005129 title "A subcomplex of RNA polymerase III subunits involved in transcription termination and reinitiation" @default.
• W2101005129 cites W1566363113 @default.
• W2101005129 cites W1574761466 @default.
• W2101005129 cites W1581154961 @default.
• W2101005129 cites W1845284873 @default.
• W2101005129 cites W1911208996 @default.
• W2101005129 cites W1966072209 @default.
• W2101005129 cites W1975593876 @default.
• W2101005129 cites W1982236741 @default.
• W2101005129 cites W1984783168 @default.
• W2101005129 cites W1987568156 @default.
• W2101005129 cites W1988904557 @default.
• W2101005129 cites W1989181646 @default.
• W2101005129 cites W1998385390 @default.
• W2101005129 cites W2003048585 @default.
• W2101005129 cites W2018289835 @default.
• W2101005129 cites W2027727311 @default.
• W2101005129 cites W2035225194 @default.
• W2101005129 cites W2037852650 @default.
• W2101005129 cites W2039635489 @default.
• W2101005129 cites W2044853699 @default.
• W2101005129 cites W2053177462 @default.
• W2101005129 cites W2054084236 @default.
• W2101005129 cites W2056482513 @default.
• W2101005129 cites W2060097540 @default.
• W2101005129 cites W2072017376 @default.
• W2101005129 cites W2075341922 @default.
• W2101005129 cites W2076195266 @default.
• W2101005129 cites W2091007136 @default.
• W2101005129 cites W2094778829 @default.
• W2101005129 cites W2100837269 @default.
• W2101005129 cites W2107324422 @default.
• W2101005129 cites W2152737612 @default.
• W2101005129 cites W2158364267 @default.
• W2101005129 cites W2165096870 @default.
• W2101005129 cites W2169254909 @default.
• W2101005129 cites W2169333620 @default.
• W2101005129 cites W2170043976 @default.
• W2101005129 cites W2171526912 @default.
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