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Facilitated Recycling Pathway for RNA Polymerase III

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Facilitated Recycling Pathway for RNA Polymerase III View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Cell, Vol. 84, 245±252, January 26, 1996, Copyright 1996 by Cell Press Facilitated Recycling Pathway for RNA Polymerase III Giorgio Dieci* and Andre Sentenac exception of TFIID, and a new complex must be reas- Service de Biochimie et Ge ne tique Mole culaire sembled for each new cycle (Zawel et al., 1995). TFIID Commissariat aÁ l'Energie Atomique±Saclay remains promoter-bound through the transcription cycle F-91191 Gif-sur-Yvette Cedex and therefore facilitates reinitiation on the same tem- France plate (Hawley and Roeder, 1987; Jiang and Gralla, 1993). However, the release and reassociation of the other fac- tors make the reinitiation process more complex than Summary in the case of pol III and thus increase the number of critical checkpoints for transcriptional modulation. We show that the high in vitro transcription efficiency Some activators of pol II transcription have indeed been of yeast RNA pol III is mainly due to rapid recycling. shown to be required at each new cycle (Arnosti et al., Kinetic analysis shows that RNA polymerase recycling 1993; Roberts et al., 1995). Reinitiation on class III genes, on preassembled tDNA·TFIIIC·TFIIIB complexes is which bypasses almost all the steps required for the much faster than the initial transcription cycle. High initial transcription cycle, does not allow such a fine efficiency of RNA pol III recycling is favored at high regulation, but potentially leads to more efficient RNA UTP concentrations and requires termination at the production. natural termination signal. Runoff transcription does Pol III-transcribed genes are small and can efficiently not allow efficient recycling. The reinitiation process direct multiple rounds of transcription in vitro (Wolffe et shows increased resistance to heparin as compared al., 1986; Kassavetis et al., 1989). This efficiency could with the primary initiation cycle, as if RNA polymerase be accounted for, at least in part, by the extreme stability was not released after termination. Indeed, template of the TFIIIB·DNA complex, which does not dissociate competition assays show that RNA pol III is committed after transcription initiation (Kassavetis et al., 1990). to reinitiate on the same gene. A model is proposed However, previous kinetic analyses of open complex where the polymerase molecule is directly transferred formation (Kassavetis et al., 1992b) and overall initiation from the termination site to the promoter. (Dieci et al., 1995a) by yeast RNA pol III on preformed preinitiation complexes showed that these processes Introduction are relatively slow. It was difficult to imagine how itera- tion of slow processes could account for a high pol The central event in the activation of class III genes is the III reinitiation frequency. In the present work, we have assembly of stable preinitiation complexes containing explored the process of reinitiation to solve this intri- transcription factors IIIC and IIIB (TFIIIC and TFIIIB). guing paradox. TFIIIC specifically binds to tRNA genes by recognizing Our analysis revealed an unexpected property of the their intragenic promoter elements. Bound TFIIIC then class III transcription machinery that is responsible for specifically recruits TFIIIB on the 59-flanking region of its high reinitiation efficiency. After the first initiation class III genes, and TFIIIB, in turn, recruits RNA polymer- event on a given preinitiation complex, RNA pol III be- ase III (pol III) (Geiduschek and Kassavetis, 1992). In comes committed to more rapidly transcribing the same yeast, TFIIIB is constituted of TATA box±binding protein gene in a way that is termination dependent. The general (TBP) and at least two other components of 70 and 90 implications of such a mechanism for the control of gene kDa (Kassavetis et al., 1992a). Yeast TFIIIB does not expression are discussed. appear to be a stable molecular entity (Huet et al., 1994), and in vitro, its recruitment on class III genes follows a Results multistep pathway involving the sequential addition of protein components to the template (Kassavetis et al., Kinetics of Initiation and Reinitiation 1992a). Once established, the TFIIIB±DNA interaction is by Yeast RNA Pol III highly stable in vitro under conditions that dissociate Free RNA pol III needs several minutes to complete open TFIIIC (Kassavetis et al., 1990). TFIIIB is alone responsi- complex formation on a preformed tDNA·TFIIIC·TFIIIB ble for the recruitment of pol III at the transcription start preinitiation complex (Kassavetis et al., 1992b), and we site. Even after stripping off TFIIIC, it can direct multiple recently observed a similar time course for the initiation rounds of transcription by pol III (Kassavetis et al., 1990). of RNA synthesis (Dieci et al., 1995a). The time course Therefore, after the slow step of preinitiation complex of initiation by yeast pol III on a preinitiation complex is assembly, a class III gene remains stably committed to shown in Figure 1A. Stable preinitiation complexes were transcription during the following reinitiation events, in formed by preincubation of SUP4 tDNA with TFIIIC and which pol III is the only recycling factor. The situation TFIIIB components; then RNA pol III was added together seems to be different in the case of RNA pol II. All the with ATP, CTP, and 32P-labeled UTP. In the absence of components of a pol II preinitiation complex dissociate GTP, a halted stable ternary complex is formed, con- during the initiation-to-elongation transition, with the taining a 17 nt nascent RNA (Kassavetis et al., 1989). Under the experimentalconditions used, RNA chain initi- *Permanent address: Istituto di Scienze Biochimiche, UniversitaÁ di ation, as monitored by the formation of the 17-mer prod- Parma, Viale delle Scienze, I-43100 Parma, Italy. uct, was completed in 5 min, with a t1¤2 of about 2 min. Cell 246 in single and multiple round transcription reactions, as described by Kassavetis et al. (1989). Note that this one and all subsequent transcription assays were carried out at limiting pol III concentration, to be sure that reinitiation was performed by the same pol III molecules that first initiated (see Experimental Procedures). Stable preinitia- tion complexes (tDNA·TFIIIC·TFIIIB) were formed; then limiting pol III was added together with ATP, CTP, and UTP, to form ternary complexes containing a 17 nt na- scent RNA. These complexes were completely resistant to 0.3 mg/ml heparin (which abolishes reinitiation), as shown by the fact that the 17 nt RNA could be quantita- tively chased to full-length RNA in the presence of this heparin concentration (Kassavetis et al., 1989; Dieci et al., 1995a; data not shown). RNA synthesis was allowed to resume by the addition of GTP, in the presence of heparin (single-round transcription) or in its absence (multiple-round transcription). The multiple-round ver- sus single-round transcript ratio corresponded to 4.5, 7, and 9 new rounds of transcription after, repectively, 2, 4, and 6 min. This corresponded to an average of 35 s per transcription cycle, a value in good agreement with previous estimations (Kassavetis et al., 1989). Such a high efficiency of reinitiation was in marked contrast with the relatively slow rate of the primary initiation pro- cess. On stable preinitiation complexes, recycling pol III completed each new cycle 5- to 10-fold more rapidly than the first round of transcription. Figure 1. Transcription Initiation and Reinitiation on the SUP4 tRNA The efficiency of reinitiation was generally found to Gene be influenced by concentration of nucleoside triphos- (A) Overall time course of initiation by pol III. A stable preinitiation phates (NTPs). Varying concentration of UTP had the complex was formed onthe SUP4 tRNA gene as described in Experi- most dramatic effect. As shown in Figure 1C, lowering mental Procedures. Pol III (50 ng) was then added together with UTP concentration from 100 mMto20mM did not ATP (500 mM), CTP (500 mM) and [a-32P]UTP (10 mM; 40 mCi/nmol). change, as expected, the output of a single-round tran- At the indicated times, samples were taken from the mixture and transferred to tubes chilled in dry ice to stop the reaction. The scription reaction (compare lanes 3 and 1), nor did it reaction products were separated on a 15% polyacrylamide, 7 M alter the kinetics of initiation by free RNA pol III (data urea gel. The position of the 17 nt transcript is indicated. The upper not shown), but it did decrease the efficiency of reinitia- part of the figure shows a scheme of the incubation protocol. tion by a factor of 2 to 3 (compare lanes 4 and 2). (B) Time course of pol III recycling. A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Initiation and Reinitiation by Pol III Have Procedures. Pol III (50 ng) was then added together with ATP (500 Different Heparin Sensitivities mM), CTP (500 mM), and [a-32P]UTP (100 mM; 4 mCi/nmol), and the To try to differentiate the processes of initiation and incubation was continued for 10 min. Transcription was restarted by the addition of GTP (500 mM) in the presence (lanes 1 and 2) or reinitiation, we explored their sensitivity to heparin treat- absence (lanes 3±5) of heparin (0.3 mg/ml). At the indicated times, ment (Figure 2). When free RNA pol III was added to samples were taken from the mixtures and transferred to tubes preinitiation complexes together with heparin, ATP, chilled in dry ice to stop the reaction. The products were separated CTP, and UTP, heparin almost totally inhibited 17-mer on a 6% polyacrylamide, 7 M urea gel. The position of the tRNA synthesis at a concentration of 2 mg/ml, and half-inhibi- gene primary transcript (SUP4 tRNA) is indicated.
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