Facilitated Recycling Pathway for RNA Polymerase III

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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 a` 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 factors 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 iteration 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 5Ј-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, Universita` 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 preinitiation 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 nascent 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 versus 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 process. 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 ␮Mto20␮M did not ATP (500 ␮M), CTP (500 ␮M) and [␣-32P]UTP (10 ␮M; 40 ␮Ci/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 ␮M), CTP (500 ␮M), and [␣-32P]UTP (100 ␮M; 4 ␮Ci/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 ␮M) 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 ␮g/ml, and half-inhibi- gene primary transcript (SUP4 tRNA) is indicated. S, single-round tory concentration was 0.25–0.5 ␮g/ml (Figure 2A). On transcription; M, multiple-round transcription. (C) UTP dependence of recycling. Transcription reactions were as the other hand, when RNA pol III is engaged in transcrip- described for (A), with the exception that UTP concentration was tion elongation, it becomes resistant to high heparin varied as indicated. To keep the specific radioactivity constant, concentrations. Thus, a concentration of 300 ␮g/ml is reactions carried out at 20 ␮M, 100 ␮M, and 500 ␮M UTP contained, normally used to allow single-round transcription (Kas- respectively, 2, 10, and 50 ␮Ci of [␣-32P]UTP. After the addition of savetis et al., 1989). To investigate the heparin sensitivity GTP only (lanes 2, 4 and 6; M, multiple-round transcription) or GTP of recycling RNA polymerase molecules, ternary com- plus heparin (lanes 1, 3 and 5; S, single-round transcription), the plexes were preformed (containing a 17-mer RNA); then, incubation was continued for 3 min. Products in lanes 2, 4, and 6 elongation and recycling (5 min) were allowed to pro- corresponded to 2.5, 5.8, and 7.0 rounds of transcription, respectively. ceed in the presence of varying heparin concentrations (Figure 2B). Under these conditions, 50% inhibition was obtained at 5 ␮g/ml, and a concentration of 100 ␮g/ml The time course of the first initiation event contrasted was required to prevent reinitiation completely. The re- with the average duration of one transcription cycle un- sidual transcription activity at 100–500 ␮g/ml corre- der reinitiation conditions, as determined by the experi- sponded, as expected, to heparin-resistant, single- ment shown in Figure 1B. The average time for one round transcription. Initiating pol III is thus at least transcription cycle by recycling RNA pol III was esti- 10-fold more sensitive to heparin than recycling RNA mated by comparing the amount of transcript produced polymerase molecules. Facilitated RNA Polymerase Recycling 247

Figure 3. Gene Commitment by Recycling RNA Pol III (A) The tRNALeu3 and SUP4 tRNA genes were separately preincubated with TFIIIC and reconstituted TFIIIB, to allow for the formation of stable preinitiation complexes. Pol III (50 ng) was then added to one of the two complexes (template 1) together with ATP (500 M), Figure 2. Heparin Inhibition of Initiation and Reinitiation ␮ CTP (500 ␮M), UTP (100 ␮M), and [␣-32P]UTP. For the reaction in (A) Heparin sensitivity of initiation. A stable preinitiation complex lane 5, pol III (50 ng) was added to a mixture of the two preinitiation was formed on the SUP4 tRNA gene as described in Experimental complexes. The incubation was continued for 10 min, and then GTP Procedures. Pol III (50 ng) was then added together with ATP (500 (500 ␮M) was added alone (lanes 3–5), together with heparin (lanes ␮M), CTP (500 ␮M), [␣-32P]UTP (10 ␮M; 40 ␮Ci/nmol), and variable 1 and 2), or together with the competitor template preassembled in concentrations of heparin, as indicated. Synthesis of the 17-mer a stable preinitiation complex (template 2; lanes 6 and 7). Transcrip- was allowed to proceed for 15 min. The products were separated tion was allowed to proceed for 5 min, and then reactions were on a 15% polyacrylamide, 7 M urea gel. The position of the 17 nt blocked and the products separated on a 6% polyacrylamide, 7 M transcript is indicated. urea gel. The position of the two primary transcripts is indicated. (B) Heparin sensitivity of reinitiation. A stable preinitiation complex Reactions in lanes 1–4 contained 20 ␮Ci of [␣-32P]UTP in a final was formed on the SUP4 tRNA gene as described in Experimental volume of 50 ␮l. Reactions in lanes 5–7 contained 40 ␮Ci of Procedures. Pol III (50 ng) was then added together with ATP (500 [␣-32P]UTP in a final volume of 100 ␮l. S, single-round transcription; ␮M), CTP (500 ␮M) and [␣-32P]UTP (100 ␮M; 4 ␮Ci/nmol) and the M, multiple-round transcription. The ratios between SUP4 and Leu3 incubation continued for 10 min. GTP was then added, together transcription were 1.7, 7.0, and 0.3 in lanes 5, 6, and 7, respectively. with variable concentrations of heparin, as indicated. The reactions (B) Lanes 1 and 3, single-round transcription reactions done as in were stopped after 5 min (equivalent to about eight rounds of tran- lanes 1 and 2 of (A), respectively. Lanes 2 and 4, SUP4 tDNA or scription in the absence of heparin) and the products separated on tDNALeu3 were preincubated with TFIIIC and TFIIIB to form stable a 6% polyacrylamide, 7 M urea gel. The position of the tRNA gene preinitiation complexes, then added to a mixture containing pol III primary transcript is indicated. (50 ng) and all four NTPs, at the same concentrations as in (A), and multiple rounds of transcription were allowed for 5 min. The ratios between the signals in lanes 2 and 4 and the signals of correspond- Gene Commitment of Recycling RNA Pol III ing single-round transcription reactions were 5.6 and 5.0 for the SUP4 and Leu3 genes, respectively. The above results suggested that, once RNA pol III has initiated, it may rapidly reinitiate on the same template, possibly without being released at each cycle. If this rounds of transcription were allowed, pol III equally tran- were the case, a recycling pol III would be committed scribed the two templates (lane 5), indicating that tran- to transcribe the same gene. To explore a possible gene scription of neither gene was intrinsically favored. In commitment, recycling pol III was given the choice be- lanes 6 and 7, pol III was first sequestered in ternary tween two genes (Figure 3A). The tRNALeu3 and SUP4 complexes on template 1 (either SUP4 or Leu3 gene), tRNATyr genes were chosen, because their primary tran- and then multiple rounds of transcription were allowed scription products have different electrophoretic mobili- to proceed for 5 min in the presence of an equimolar ties. In addition, stable ternary complexes can be formed amount of template 2 preassembled into preinitiation on both genes in the presence of the same subset of complex with TFIIIC and TFIIIB. In both cases, pol III did nucleotides, ATP, CTP, and UTP (Kassavetis et al., not redistribute itself equally on both genes, as would be 1989). Pol III efficiently recycled on both genes (Figure expected if it had been released after the first cycle. 3A; compare lanes 1 and 2 with lanes 3 and 4, respec- Instead, pol III preferentially recycled on template 1, tively). The reinitiation frequency (i.e., the multiple-round while transcription of the competitor template 2 was versus single-round transcript ratio) on tDNALeu3 (6 cy- significantly excluded. Figure 3B additionally shows that cles/5 min) was slightly lower than on tDNA SUP4 (8 when template 1 was omitted, template 2 was much cycles/5 min). When a limiting amount of pol III was more actively transcribed, during 5 min of incubation, added to a mixture of the two genes (each being preas- than when pol III had initiated on template 1 prior to sembled into preinitiation complexes) and then multiple template 2 addition (as in lanes 6 and 7 of Figure 3A). Cell 248

Figure 5. Time Course of Initiation ona Previously Transcribed Tem- plate A stable preinitiation complex was formed on the SUP4 tRNA gene as described in Experimental Procedures. The incubation mixture was split into two parts. One part received pol III (50 ng) together Figure 4. Time Course of 17-mer Synthesis by a Complete Initiation with ATP (500 M), CTP (500 M), and UTP (100 M); after a 10 min Complex ␮ ␮ ␮ incubation, GTP (500 ␮M) was added together with heparin (0.25 The experiment was as in Figure 1A, but pol III alone was preincu- mg/ml), to generate preinitiation complexes transcribed once (Kas- bated 10 min with the preinitiation complex before the addition of savetis et al., 1990). The other half of the mixture, containing never- ATP, CTP, and UTP. A scheme of the incubation protocol is shown transcribed complexes, received only heparin. Each of the two mix- at the top. tures (110 ␮l) was loaded onto a 1 ml Sepharose CL-2B gel filtration column. Fractions 9–11 from each column, containing stablepreiniti- ation complexes but not free RNA polymerase (data not shown), On the basis of all these data, we conclude that recy- were collected and pooled. Pol III (50 ng) was then added to each cling RNA pol III is predominantly committed to the first pool, together with ATP (500 ␮M), CTP (500 ␮M), and [␣-32P]UTP transcribed gene. (10 ␮M; 40 ␮Ci/nmol), and samples of the reactions were blocked at the indicated times. The reaction products were separated on a Mechanisms of Facilitated Reinitiation 15% polyacrylamide, 7 M urea gel. The position of the 17 nt tran- by RNA Pol III script is indicated. Reinitiation could proceed faster if one or more rate- limiting steps of initiation are performed at a higher rate treated with heparin and gel-filtrated in the absence of in the following rounds. The kinetics of initiation shown in RNA polymerase. As shown in Figure 5, initiation on Figure 1A are determined by the rate of three successive previously transcribed preinitiation complexes pro- steps: recruitment of pol III, DNA melting, and initiation ceeded with a t1⁄2 comprising between 1 and 2 min. After of RNA synthesis. The contribution of the latter step, 2 min, about 70% of the complexes had initiated in i.e., the time required for the formation of the 17-mer this case (Figure 5, bottom), and 50% in the case of RNA, was determinedby the experiment shown in Figure preinitiation complexes that had not been previously 4. RNA pol III was allowed to associate with tDNA• transcribed (Figure 5, top). This slight increase in initia- TFIIIC•TFIIIB complexes for 10 min at 22ЊC before the tion rate on previously transcribed complexes, which addition of ATP, CTP, and UTP. Under these conditions, was reproducibly observed in several experiments, 17-mer synthesis was completed within 10 s (Figure could not account by itself for the fast reinitiation rate 4). Initiation by free RNA pol III thus involves a slow observed in Figures 2B and 2C. We have not explored association or DNA-opening step, followed by rapid further the basis for this small increase in initiation effi- RNA chain initiation. The uncoupling of initiation and ciency. It should be noted, however, that transient modi- reinitiation rates is most likely due to the ability of recy- fications of the preinitiation complex would likely be lost cling pol III to carry out this slow step at a higher rate. during heparin treatment followed by gel filtration. Thus, The use of limiting amounts of RNA polymerase made we can only conclude that facilitated recycling does unlikely the possibility that fast reinitiation was simply not involve long-lived and stable modifications of the due to a rapid recruitment of excess, DNA-bound en- preinitiation complex. zyme molecules. This was confirmed by the observation that facilitated reinitiation also occurred on very short The Pol III Terminator Sequence Is DNA templates such as a 270 bp DNA fragment harbor- Required for Efficient Reinitiation ing the SUP4 tRNA gene (data not shown). Since termination and RNA release immediately precede Facilitated reinitiation could proceed through a modi- reinitiation, the modality of termination by pol III could fication of preinitiation complexes, occurring during the influence reinitiation efficiency. In class III genes, the first transcription cycle, that would increase the rate termination signal is a run of T residues in the nontran- of subsequent reinitiation cycles. This point was ad- scribed strand of DNA (Geiduschek and Tocchini-Valen- dressed by measuring the time course of de novo RNA tini, 1988). To examine the role of this signal in facilitated chain initiation by free pol III on a template that had been reinitiation, we compared the recycling efficiency on a through one transcription cycle. Preinitiation complexes linear template with or without a functional terminator. were formed, subjected to single-round transcription We chose for this purpose the truncated form of the in the presence of heparin, isolated by gel filtration to yeast U6 gene lacking the extragenic B box (Brow and remove RNA polymerase, and reused for RNA chain Guthrie, 1988). Transcription of this gene in a purified initiation. As a control, preinitiation complexes were system does not require TFIIIC and is only dependent on Facilitated RNA Polymerase Recycling 249

Figure 6. Requirement of the Terminator Element for Efficient Reini- tiation on the U6 Gene (A) Decreased reinitiation frequency after runoff termination. TFIIIB Figure 7. Model for the Pathways of Transcription Reinitiation by was assembled on the truncated version of the yeast U6 gene (con- RNA Pol III tained in pTaq6 plasmid) as described in Experimental Procedures. Reactions in lanes 1 and 2 contained the uncut, circular plasmid. For simplicity, TFIIIC has been omitted from the scheme. Steps 1 For reactions in lanes 3 and 4, the plasmid was linearized at the and 2 summarize a simple transcription cycle, from initiation by free polylinker SmaI site, downstream of the U6 terminator sequence. pol III to termination. Step 1 can be inhibited by 2 ␮g/ml heparin. For reactions in lanes 5 and 6, the plasmid was linearized at the Concomitant with the termination step, two alternative pathways EcoNI site in the coding region, 30 bp upstream of the terminator can be followed by pol III: it can either dissociate (step 3b) and then (Moenne et al., 1990). Pol III (50 ng) was added together with CTP slowly reinitiate a new cycle from step 1, or undergo a termination- (500 ␮M), GTP (500 ␮M), and [␣-32P]UTP (200 ␮M; 2 ␮Ci/nmol), and coupled, fast reinitiation event without being released from the tem- the incubation was continued for 15 min to allow for the formation of plate (steps 3a and 4). The latter pathway can take place in the a ternary transcription complex containing a 7 nt nascent transcript presence of 2 ␮g/ml heparin and can be inhibited by 50–100 ␮g/ml (Brow and Guthrie, 1988). ATP was then added in the presence heparin. A single arrow in the figure does not imply that there is (lanes 1, 3, and 5) or the absence (lanes 2, 4, and 6) of heparin only one biochemical step involved. For details, see Discussion. to inhibit reinitiation, and the incubation was continued for 6 min. Reaction products were separated on a 6% polyacrylamide, 7 M urea gel. The positions of the complete and runoff U6 RNA gene 5) was about half the signal produced by a single round products are indicated. S, single-round transcription; M, multiple- of complete transcription (lane 3), but this was expected, round transcription. because the runoff transcript contains half as many Us (B) The terminator sequence does not influence the affinity of the preinitiation complex for RNA pol III. TFIIIB was separately assem- as the complete U6 transcript. When pol III was incu- bled on SmaI- and EcoNI-linearized pTaq6, and then pol III was bated with a mixture of the two linear templates (each added to a mixture of the two templates, together with CTP (500 preassembled with TFIIIB), the same number of single- ␮M), GTP (500 ␮M), and [␣-32P]UTP (200 ␮M; 2 ␮Ci/nmol). After 15 round transcripts was produced from the two templates, min, ATP (500 ␮M)was added with (lane 1) or without (lane 2) heparin, indicating that limiting pol III had been equally recruited and transcription was allowed to proceed for 6 min in a final volume by the two genes (Figure 6B, lane 1). The terminator of 100 ␮l. Reaction products were separated on a 6% polyacrylamide, 7 M urea gel. The positions of the complete and runoff U6 element hasthus no influence on the affinity of the preini- RNA gene products are indicated. S, single-round transcription; M, tiation complex for pol III. However, RNA polymerase multiple-round transcription. molecules reinitiated much more efficiently on the terminator-containing template than on the terminator-defi- the upstream assembly of TFIIIB (Moenne et al., 1990). It cient gene (lane 2). In experiments not shown, a circular is thus possible to obtain runoff transcription from the pTaq6 template preassembled with TFIIIB was added U6 promoter by linearizing the template at the EcoNI to a reaction in which pol III had performed one cycle site located in the coding region, 30 bp upstream of the of runoff transcription. These pol III molecules could terminator (Moenne et al., 1990). The pTaq6 plasmid initiate and reinitiate transcription on pTaq6 template was linearized at the unique EcoNI (runoff transcription) with the same efficiency as freshly added pol III. This or SmaI (normally terminated transcription) sites, and result indicated that the reduced recycling ability on the recycling ability of pol III on these templates was the U6 runoff template was not due to the artifactual tested by comparing single- and multiple-round tran- inactivation of pol III by runoff transcription. scription reactions (Figure 6). Linearization downstream of the terminator region did not affect the reinitiation Discussion efficiency compared with that of a circular template: during 6 min of recycling, about six cycles of transcrip- The analysis reported here reveals a new mechanism tion were performed by pol III on both templates (Figure allowing a high level of RNA polymerase recycling after 6A; compare lanes 3 and 4 with lanes 1 and 2). In con- a single gene activation event. We show that pol III trast, linearization upstream of the terminator strongly preferentially recycles on the same template, in a way reduced transcription reinitiation (compare lanes 5 and that allows it to complete new cycles more rapidly than 6): fewer than two cycles were performed on this tem- the initial one. This conclusion is summarized in the plate during the same period of time. The signal corre- model presented in Figure 7. sponding to a single round of runoff transcription (lane In the first step of initiation (step 1 in Figure 7), free Cell 250

RNA pol III is recruited by a stable tDNA•TFIIIC•TFIIIB The increased heparin resistance of recycling pol III preinitiation complex to form a closed complex that, at is probably not restricted to the case of the yeast pol room temperature, easily isomerizes to an open complex III system. In their study of the effects of sarkosyl on (Kassavetis et al., 1992b). This overall slow process of human pol III transcription, Kovelman and Roeder (1990) RNA polymerase recruitment and open complex forma- found a sarkosyl concentration (0.015%) that inhibited tion is very sensitive to heparin. When pol III has been a pol III–dependentstep in the first round of transcription allowed to associate with the preinitiation complex in of the VA1 RNA gene, but that did not inhibit reinitiation. the absence of NTPs, the resulting complex is able to It is thus tempting to speculate that reinitiation by human initiate RNA synthesis very rapidly, in less than 10 s (step pol III also proceeds through the facilitated pathway. 2 in Figure 7), and becomes resistant to high heparin Facilitated reinitiation by pol III could involve a specific concentrations (Kassavetis et al., 1989). After the elon- contact between terminating pol III and a component gation phase, which is very rapid on the short tRNA of the preinitiation complex. As represented in the model genes (Matsuzaki et al., 1994), a pol III molecule must in Figure 7, this contact could involve DNA looping, undergo the termination process, which includes, as facilitated by the short size of the transcribed region, potentially separate steps, the release of the terminated and may require a conformational change of the pol III RNA chain and the release of RNA polymerase itself molecule. It has been recently shown that the oligo(dT) from the template (Chamberlin, 1974). These steps have tract of Escherichia coli transcription terminators can the potential to influence strongly the ability of polymer- induce a conformational change in E. coli RNA polymerase to reinitiate. The common view of reinitiation is that ase by acting as an inchworming signal (Nudler et al., RNA polymerase needs to be released in order to be 1995). The pol III terminator element could be required to recruited by preinitiation complexes to start a new tran- give pol III the proper conformation for the interactions scription cycle (step 3b in Figure 7). We propose that, leading to facilitated reinitiation. Campbell and Setzer in the case of pol III, a preferential termination pathway (1992) have shown that termination signal recognition allows RNA release and transcription reinitiation to be by pol III can be experimentally uncoupled from poly- performed without release of RNA polymerase (steps merase and transcript release. Pausing of pol III at the 3a and 4 in Figure 7). This pathway leads to an increased termination signal before release would give time for the initiation rate and to a distinctive heparin resistance. facilitated reinitiation event to take place; on the other The marked dependence of the reinitiation rate on NTP hand, runoff termination would directly lead to the re- and, in particular, UTP concentration (Figure 1C) can lease of elongating polymerase molecules. Some re- be easily explained in the context of this pathway: the ports have indicated that the RNA-binding protein La initiation phase being no longer rate-limiting, the overall stimulates human pol III transcription by facilitating mul- transcription process can be significantly stimulated by tiple rounds of transcription initiation (Gottlieb and the UTP-induced increase in the rates of elongation and Steitz, 1989a, 1989b; Maraia et al., 1994). Even if efficient termination. The terminator element appears to be re- class III gene transcription has been reconstituted from quired for pol III to enter the facilitated initiation pathway, homogeneous or highly purified components (Kassa- since runoff termination leads to slow reinitiation (Figure vetis et al., 1992a; Joazeiro et al., 1994), the possibility 6), probably owing to spontaneous pol III dissociation cannot be excluded that a yeast La homolog (Yoo and (as in step 3b of Figure 7). Wolin, 1994; Lin-Marq and Clarkson, 1995), or another The existence of a pathway avoiding dispersion of factor, might play a role in the facilitated reinitiation recycling pol III into the free RNA polymerase pool is pathway. supported by the finding that, during multiple rounds of There are noticeable differences in the sensitivity of initiation, pol III is committed to the gene on which it pol II and pol III transcription systems to inhibitors such has first initiated (Figure 3). By template exclusion as heparin or sarkosyl, which most likely reflect differ- assays and glycerol gradient sedimentation of transcrip- ences in reinitiation mechanisms. In the case of pol II tion complexes, Jahn et al. (1987) previously provided transcription, reinitiation is inhibited by the same sarko- some evidence that human pol III is retained in the origi- syl concentration (0.025%) that inhibits initiation com- nal transcription complex during the normal reinitiation plex assembly (Hawley and Roeder, 1987). This indi- cycle. Our data are in agreement with those of Jahn et cates that, in contrast with what was observed with pol al., even if, in this case, the extent of reinitiation was III, the sarkosyl-sensitive step in pol II initiation has to not evaluated, but they are at variance with the conclu- be repeated at each reinitiation cycle. Zawel et al. (1995) sions of Bieker et al. (1985), who suggested that human recently showed that, following the transition from initia- pol IIIequilibrates during multiple rounds of transcription tion to elongation, only TFIID remains promoter-bound, on different 5S RNA templates. In the template exclusion whereas TFIIB, TFIIE, TFIIF, and TFIIH are released and assays of Bieker et al., however, transcription was al- must reassociate at each cycle. The need for a partial lowed to proceed for long periods of time. At each cycle, iteration of preinitiation complex assembly could explain a small percentage of pol III molecules can be released the sarkosyl sensitivity of pol II reinitiation. On the other from the first gene (as in step 3b of Figure 7) and can hand, the fact that TFIID is left behind on the template therefore initiate transcription on the second template. after the first round of initiation can explain the uncou- Increasing the number of cycles will eventually lead to pling of the rates of initiation and reinitiation that has a complete redistribution of pol III. It should be noted been observed for GAL–AH-dependent pol II in vitro that, under our short incubation conditions, pol III com- transcription (Jiang and Gralla, 1993). mitment was not absolute (see Figure 3). In the case of pol III, the reinitiation process seems Facilitated RNA Polymerase Recycling 251

to have gained its maximal efficiency: not only is a func- single-round transcription was from Sigma (H-2149 type). Reaction tional preinitiation complex constantly left at the pro- products were purified by phenol extraction and double ethanol moter, but, in addition, the polymerase can initiate new precipitation and separated on 0.8 mm 6% or 15% polyacrylamide gels (20:1 [w/w], acrylamide:bisacrylamide) containing 7 M urea. cycles more rapidly than the first one without being Transcripts were quantitated by a PhosphorImager with Image completely released. This optimization of transcript pro- Quant software (Molecular Dynamics). The number of transcripts duction after a single gene activation event is probably produced in a single-round transcription reaction was determined essential to satisfy the high cellular needs for class III by comparison, on the same gel, of the radioactive signal produced gene products. However, a facilitated reinitiation path- by single-round transcripts with the signal of a known amount of a 32 way might also exist, in variant forms, in the other poly- P-labeled 200 bp DNA fragment of known specific radioactivity. A single round of transcription produced 8 fmol of SUP4-specific merase systems. Since rDNA transcription by RNA pol transcript. Therefore, only 10% of the template molecules could be I must also be highly efficient, it would not be surprising assembled into active transcription complexes. This was expected, if the pol I transcription system had features allowing because in our assay, transcription components are limiting with facilitated reinitiation to take place. Indeed, the tandem respect to template DNA. Indeed, increasing the amounts of tran- arrangement of eukaryotic rRNA genes has led to the scription components without changing the DNA concentration re- hypothesis that a specialized mechanism may exist for sulted in proportional increase of active transcription complexes (data not shown). the direct transfer of pol I from the terminator to the Ternary complex purification was carried out as previously de- adjacent promoter without dissociation (Baker and Platt, scribed (Dieci et al., 1995a). 1986; Johnson and Warner, 1989). Mechanisms allowing RNA pol II to terminate and reinitiate without being dis- Acknowledgments persed into the free enzyme pool are also possibly used in situations where a maximal level of transcription is Correspondence should be addressed to A. S. We thank Janine required. Huet for the gift of pure RNA pol III and TBP; Michel Werner, Chris- tophe Carles, Olivier Lefebvre, and Marie-Claude Marsolier for help- Experimental Procedures ful discussions and critical comments on the manuscript; and Carl Mann for improving the text. G. D. was supported by a long-term European Molecular Biology Organization Postdoctoral Fellowship. Purification of RNA Pol III and Class III This work was supported by Biotechnology Programme Grant B102- Transcription Factors CT92-0090 from the European Community. Purification of transcription proteins was carried out following previously described procedures. Pure RNA pol III (a gift of J. Huet) Received September 13, 1995; revised October 30, 1995. was prepared as described by Huet et al. (1985). Affinity-purified TFIIIC was prepared as described by Gabrielsen et al. (1989). TFIIIB activity was reconstituted from the three subcomponents TBP, References TFIIIB70, and fraction BЈЈ. Yeast recombinant TBP, expressed in E. coli from plasmid pET3b(rTFIIDY) (a gift from J.-M. Egly), was purified Arnosti, D.N., Merino, A., Reinberg, D., and Schaffner, W. (1993). by the procedure of Burton et al. (1991). Recombinant histidine- Oct-2 facilitates functional preinitiation complex assembly and is tagged TFIIIB70 was expressed in E. coli from plasmid pSH360 (a continuously required at the promoter for multiple rounds of tran- gift from S. Hahn; Colbert and Hahn, 1992) and purified by chroma- scription. EMBO J. 12, 157–166. tography on Ni2ϩ–NTA–agarose (Qiagen) under native conditions, Baker, R.E., and Hall, B.D. (1984). Structural features of yeast tRNA as described (Dieci et al., 1995b). Fraction BЈЈ was extracted from genes which affect transcription factor binding. EMBO J. 3, 2793– chromatin pellets and partially purified on BioRex 70 (Bio-Rad) ac- 2800. cording to the protocols of Kassavetis et al. (1992a). Baker, S.M., and Platt, T. (1986). Pol I transcription: which comes first, the end or the beginning? Cell 47, 839–840. Specific In Vitro Transcription Assays Bieker, J.J., Martin, P.L., and Roeder, R.G. (1985). Formation of a Plasmid DNAs used for in vitro transcription reactions were the rate-limiting intermediate in 5S RNA gene transcription. Cell 40, following: pRS316-SUP4, containing the yeast SUP4 tRNA gene (S. 119–127. Shaaban, personal communication); pGE2-WT, containing the yeast tRNALeu3 gene (Baker and Hall, 1984); and pTaq6, containing a trun- Brow, D.A., and Guthrie, C. (1988). Spliceosomal RNA U6 is remark- cated form of the yeast U6 RNA gene lacking the extragenic B box ably conserved from yeast to mammals. Nature 334, 213–218. (Brow and Guthrie, 1988). Transcription reactions were carried out Burton, N., Cavallini, B., Kanno, M., Moncollin, V., and Egly, J.-M. in a final volume of 50 ␮l and contained 20 mM Tris–HCl (pH 7.9), (1991). Expression in Escherichia coli purification and properties of 90 mM KCl, 5 mM MgCl2, 10% glycerol (v/v), 0.1 mM EDTA, 0.5 mM the yeast general transcription factor TFIID. Protein Exp. Purif. 2, 32 each of ATP, CTP, and GTP, variable UTP and [␣- P]UTP (Amers- 432–441. ham) concentrations, 250 ng of plasmid DNA, 50 ng of affinity- Campbell, F.E., and Setzer, D.R. (1992). Transcription termination purified TFIIIC, 40 ng of rTBP, 50 ng of rTFIIIB70, 0.4 ␮gofBЈЈ by RNA polymerase III: uncoupling of polymerase release from ter- fraction, and 50 ng of RNA pol III. Reactions programmed with the mination signal recognition. Mol. Cell. Biol. 12, 2260–2272. U6 gene did not contain TFIIIC. In all transcription reactions, RNA pol III was limiting. The limiting pol III concentration was chosen on Chamberlin, M.J. (1974). Bacterial DNA-dependent RNA polymer- the basis of titration experiments in which pol III concentration was ase. Enzymes 10, 333–374. varied under single-round transcription conditions. In these assays, Colbert, T., and Hahn, S. (1992). A yeast TFIIB-related factor involved a plateau was reached in the presence of 100 ng of pol III, and 40 in RNA polymerase III transcription. Genes Dev. 6, 1940–1949. ng of pol III was half saturating. In all transcription assays, the Dieci, G., Hermann-Le Denmat, S., Lukhtanov, E., Thuriaux, P., Wer- template was first preincubated (15 min at 22ЊC) with TFIIIC and the TFIIIB subcomponents (or with TFIIIB only, in the case of the U6 ner, M., and Sentenac, A. (1995a). A universally conserved region RNA gene) in a final volume of 42 ␮l, to allow for the formation of of the largest subunit participates in the active site of RNA polymer- stable preinitiation complexes. Pol III and subsets of NTPs were ase III. EMBO J. 14, 3766–3776. then added, to form heparin-resistant ternary complexes containing Dieci, G., Duimio, L., Peracchia, G., and Ottonello, S. (1995b). Selec- a nascent RNA chain. Transcription was eventually allowed to re- tive inactivation of two components of the multiprotein transcription sume by the addition of the missing NTP under single- or multiple- factor TFIIIB in cycloheximide growth-arrested yeast cells. J. Biol. round transcription conditions (Kassavetis et al., 1989). Heparin for Chem. 270, 13476–13482. Cell 252

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