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Sarkosyl defines three intermediate steps in transcription initiation by RNA polymerase III: application to stimulation of transcription by E1A.

We used Sarkosyl to analyze steps along the pathway of transcription initiation by RNA polymerase III. Sarkosyl (0.015%) inhibited transcription when present prior to incubation of RNA polymerase III, TFIIIB, and TFIIIC with the VAI gene, whereas it had no detectable effect on initiation or reinitiation of transcription when added subsequently. The formation of the corresponding 0.015% Sarkosyl-resistant complex required the presence of TFIIIC, TFIIIB, and RNA polymerase III but not nucleoside triphosphates. The addition of 0.05% Sarkosyl after this early step selectively inhibited a later step in the preinitiation pathway, allowing a single round of transcription after nucleoside triphosphate addition but blocking subsequent rounds of initiation. This step occurred prior to initiation because nucleoside triphosphates were not required for the formation of the corresponding 0.05% Sarkosyl-resistant complex. These observations provided a means to distinguish effects of regulatory factors on different steps in promoter activation and function. Using 0.05% Sarkosyl to limit reinitiation, we determined that the E1A-mediated stimulation of transcription by RNA polymerase III resulted from an increase in the number of active transcription complexes.

The transcription of eukaryotic genes requires auxiliary factors in addition to one of the three eukaryotic RNA polymerases {Ng et al. 1979;Weil et al. 1979;Grummt 1981). In the case of genes transcribed by RNA polymerase III, two commonly-required factors, TFIIIB and TFIIIC, are necessary and sufficient for the transcription of tRNA and VA RNA genes (Segall et al. 1980;Shastry et al. 1982), whereas 5S gene transcription requires, in addition, the gene-specific factor TFIIIA (Engelke et al. 1980). Preinitiation complexes containing these factors are assembled on the internal promoters in a defined order (Lassar et al. 1983;Setzer and Brown 1985;Bieker et al. 1985): For VA and tRNA genes this is TFIIIC, then TFIIIB, and, finally, RNA polymerase III. On the VA and some tRNA genes, TFIIIC is sufficient for formation of a highly stable complex, while other tRNA genes require stabilization of TFIIIC binding by TFIIIB for stable complex formation (Lassar et al. 1983;Baker and Hall 1984;Fuhrman et al. 1984). In the case of the 5S gene, these interactions are preceded by the formation of a metastable 5S promoter-TFIIIA complex (Engelke et al. 1980;Lassar et al. 1983). In all cases the stable preinitiation complexes support multiple rounds of transcription and remain refractory to challenge by competing templates (Bogenhagen et al. 1982;Lassar et al. 1983;Fuhrman et al. 1984).
The steps and factors involved in the formation of a transcription complex are understood at various levels of detail. TFIIIA from Xenopus laevis has been purified to homogeneity (Engelke et al. 1980)and cloned (Ginsberg et al. 1984), and its interactions with the 5S promoter have been studied in detail (for review, see Geiduschek and Tocchini-Valentini 1988). On the other hand, although the promoter elements for VA and tRNA genes were analyzed in detail some time ago {for review, see Ciliberto et al. 1983), TFIIIB and TFIIIC from different organisms (i.e., yeast and human) have only recently been purified {e.g., see Klekamp and Weil 1986a;Waldschmidt et al. 1988;Cromlish and Roeder 1989;Yoshinaga et al. 1989). Consequently, there is correspondingly less information about the structure, mechanism of action, and regulation by other factors. This contrasts with our present understanding of prokaryotic RNA polymerase initiation mechanisms and regulatory factor interactions (for review, see McClure 1985).
Studies with selective transcription initiation inhibitors in relatively crude systems have defined distinct steps that can be analyzed further as purified components become available. Thus, in the case of specific initiation by RNA polymerase II, Hawley andRoeder (1985, 1987) showed that there are at least two steps in the overall pathway that are differentially sensitive to the anionic detergent Sarkosyl and that may correspond to steps defined by studies with more purified factors (Van Dyke et al. 1988;Buratowski et al. 1989). Sarkosyl has also been shown to affect transcription by RNA polymerase I (Kato et al. 1986) and RNA polymerase III (Klekamp and Weil 1986b), although its effects on individual steps in complex formation in the class III system have not been examined previously.
In this report, we show that different concentrations of Sarkosyl inhibit the formation of two distinct preinitiation complexes on the VAx gene and that Sarkosyl can be used to limit reinitiation and therefore to assay single rounds of transcription. This has allowed us to show that the E1A-mediated stimulation of VA~ transcription, previously demonstrated to be mediated by an increase in the level of a transcriptionally more active form of TFIIIC (Hoeffler et al. 1988), results from an increased number of functional transcription complexes.

O. 015 % Sarkosyl inhibits preinitiation complex formation
To determine whether any steps prior to transcription initiation by RNA polymerase III were differentially sensitive to inhibition by Sarkosyl, we employed the Sarkosyl inhibition of RNA polymerase II two-step assay protocol shown in Figure 1A. In step 1, the plasmid containing the adenovirus VAI RNA gene (pVAI) w a s preincubated with phosphocellulose fractions containing TFIIIB, TFIIIC, and RNA polymerase III. In step 2, transcription was started by the addition of NTPs. Sarkosyl was added to various final concentrations (as noted above the lanes in Fig. 1A) either before the addition of the factors to the V A I template in step 1 (lanes 1-61 or at the end of the preincubation and just before the addition of NTPs in step 2 (lanes 7-12). The results of control reactions to which no Sarkosyl was added are shown in lanes 1 and 7. The addition of Sarkosyl to a final concentration of 0.001% or 0.005% at either the beginning or the end of the preincubation had little effect on levels of transcription (cf. lanes 2 and 3 with lanes 8 and 9). On the other hand, the presence of 0.015% Sarkosyl during the preincubation abolished transcription (lane 4), while its addition after the preincubation of the gene and the factors (lane 10) allowed transcription to occur at control levels. The addition of higher concentrations of Sarkosyl at either the beginning or the end of the preincubation abolished transcriptional activity. (A very small amount of transcription can be detected in lanes 11 and 12 on long exposures of the autoradiograph. Its significance is addressed below.) This experiment demonstrates that the pathway of preinitiation complex formation by RNA polymerase III includes  (Wingender et al. 1986). To determine whether the VAI gene is required to confer resistance to 0.015% Sarkosyl, we performed the experiment diagrammed at the bottom of Figure lB. Preincubations and transcription reactions were performed as described in Figure 1A, except that for lane 14 the template was not added until the end of the preincubation. The complete absence of transcription in lane 14 demonstrates that the template is required to confer resistance to 0.015% Sarkosyl, and that the Sarkosyl-sensitive step is presumably along the normal pathway of preinitiation complex formation on the VA~ gene.
The results of additional Sarkosyl titrations (performed as in Fig. 1) are shown in Figure2. All results are expressed as a percent of control reactions to which no Sarkosyl was added. The solid lines represent the results of reactions to which Sarkosyl was added at the beginning of the preincubation (as for the left-hand panel of Fig. 1A), whereas the dashed lines represent those to which Sarkosyl was added at the end of the preincubation (as for the right-hand panel of Fig. 1A). The data are from two sets of experiments, circles being from one series and squares from another. Although there is minor (<20%) variability from experiment to experiment, it is clear that a preincubation of template and factors in the absence of NTPs led to the formation of a preinitiation complex which was resistant to disruption by 0.015% Sarkosyl.

Time course of formation of the complex resistant to O. O15 % Sarkosyl
We measured the amount of time required for formation of the 0.015% Sarkosyl-resistant complex by adding Sar-kosyl to a final concentration of 0.015% at variable times between 0 and 60 min after the beginning of a 60rain preincubation. (See the diagram at the bottom of Fig. 3.) Figure 3 shows the results of these transcription reactions expressed as a percent of the radioactivity incorporated in the reaction to which Sarkosyl was added at the end of the preincubation. The formation of this complex was a relatively slow process with a t~,~ of ~14 min. This suggests that the step sensitive to 0.015% Sarkosyl is not the first step in the formation of a transcription complex on the VA I gene, because this first step, the binding of TFIIIC to the promoter, is known from both template commitment (Lassar et al. 1983;Fuhrman et al. 1984;Bieker et al. 1985) and gel retardation (Hoeffler et al. 1988) assays to be a much faster process. Moreover, the kinetics observed here are very similar to the kinetics of formation of a rate-limiting complex on a 5S RNA gene (Bieker et al. 1985). Thus, the step blocked by 0.015% Sarkosyl may also be the ratelimiting step for the initiation of in vitro transcription on the VAI gene.

Factors required for the formation of the 0.015% Sarkosyl-resistan t complex
To determine which factors are required to form a complex resistant to inhibition by 0.015% Sarkosyl, phosphocellulose fractions containing TFIIIB or TFIIIC and Sarkosyl were added sequentially as diagrammed in Figure 4. In each case, a 60-min preincubation was followed by a 60-min transcription reaction. As in the experiment described above, the template was added at the beginning of the preincubation (0 min), and NTPs were added to start the transcription reaction (60 min). A fraction containing either TFIIIB or TFIIIC was added at the beginning of the preincubation (0 min), while the other fraction was added 30 min later. Sarkosyl was added to a final concentration of 0.015% either just before addition 100 ._.  . Time course of formation of the preinitiation complex resistant to 0.015% Sarkosyl. The addition of pVAI, fractions, KC1, MgC12, H20, NTPs, and stop mix was as described in the legend to Fig. 1, except that the total preincubation time was 60 rain. Sarkosyl was added at various times during the preincubation such that its final concentration during the transcription reaction would be 0.015%. Quantitation was performed as described in Materials and methods; levels of transcription were normalized to the sample to which Sarkosyl was added at the end of the preincubation. to .015% 60 of the second fraction (at 30 min) or just before the addition of the NTPs (at 60 min). The addition either of TFIIIC at 0 min and TFIIIB at 30 rain (lane 1) or of TFIIIB at 0 min followed by TFIIIC at 30 min (lane 4), in the absence of Sarkosyl, led to nearly normal levels of transcription; there was only a small decrease in transcription when TFIIIB was preincubated in the absence of TFIIIC (cf. lane 4 with lane 1), which may result from TFIIIB instability in the absence of TFIIIC (Lassar et al. 1983). Moreover, the addition of Sarkosyl at the end of the preincubation under these conditions (lanes 2 and 5) did not significantly inhibit transcription. On the other hand, the addition of Sarkosyl immediately prior to the addition of the second factor (at 30 min) eliminated transcriptional activity (lanes 3 and 6). Thus, the requirement of both TFIIIB and TFIIIC for the formation of the 0.015% Sarkosyl-resistant complex distinguishes it from the initial stable complex of TFIIIC and the VAx gene (Lassar et al. 1983}. We were interested in determining whether RNA polymerase III was also required to confer resistance to 0.015% Sarkosyl. Because both of the phosphocellulose fractions (0.35 M and 0.6 M steps) used in the experiment shown in Figure 4 contain RNA polymerase III in addition to TFIIIB and TFIIIC, we performed further purification of each of the components. RNA polymerase III derived from HeLa cell $100 extracts was purified over three columns, while TFIIIB and TFIIIC were separated from the RNA polymerase on DEAE-Sephadex A-25. (See Materials and methods for details.) Sarkosyl titrations performed as in Figure 2 with these more purified fractions were essentially identical to those with phosphocellulose fractions (data not shown). We employed these fractions in sequential preincubations as diag-rammed at the bottom of Figure 5. The addition of Sarkosyl after a preincubation of all components (lane 2) led to a small reduction in transcription compared to a control reaction to which no Sarkosyl had been added (lane

I I NTPs Stop
Sarkosyl ~l'komTI whereas Sarkosyl to a final concentration of 0.015% in the transcription reaction was added either just before the addition of NTPs to start transcription at t = 60' (60 rain) (lanes 2 and 5) or just before the addition of the second fraction at t = 30' (lanes 3 and 6). Incorporation of [a-a2p]GTP in lanes 2 and 5 were 55% and 51% of the respective controls (lanes 1 and 4).  Figure 5. Formation of the 0.015% Sarkosyl-resistant complex also requires RNA polymerase III. The experimental procedure was similar to that employed for Fig. 4, except that the total preincubation time was 30 min, and the fractions {DEAE-Sephadex fractions containing THIIB or TFIIIC and RNA polymerase III prepared as described in Materials and methods) were in all cases added in the order TFIIIC, THIIB, and RNA polymerase III. Sarkosyl was always added immediately prior to the addition of the NTPs or protein fraction. Incorporation of [a-a~P]GTP in lane 2 was 38% of control {lane 1). 1); this correlates well with what was observed with phosphocellulose fractions (cf. lanes 1 and 2 of Fig. 4; see figure legends for quantitation). On the other hand, the addition of Sarkosyl to 0.015% at any earlier stage of complex formation completely inhibited transcription (lanes 3-5). Thus, the formation of a 0.015% Sarkosylresistant complex required the presence of all three components known to be involved in the transcription of class III genes.

Reinitiation is limited by 0.05% Sarkosyl
Close inspection of very long exposures of the autoradiograph shown in Figure 1 revealed that low levels of transcriptional activity persisted when Sarkosyl was added to 0.05% or 0.25% at the end of the preincubation (data not shown). To investigate whether these low levels of transcription resulted from single rounds of transcription, we performed time courses of transcription as diagrammed at the bottom of Figure 6. The template and factors were preincubated as described for the previous experiments, and Sarkosyl was added to a final concentration of either 0.015% ( The data shown in Figures 1-5 imply that if reinitiation (defined as the production of more than one transcript from a particular complex) were occurring in this system, then the addition of 0.015% Sarkosyl after a preincubation did not preclude multiple rounds of transcription from active complexes, as overall transcriptional levels were not significantly changed from levels seen in the absence of Sarkosyl. The time course of transcription in the presence of 0.015% Sarkosyl ( Fig. 6A and data not shown) is consistent with this assumption, as the rate of VAI transcript production was constant for over 1 hr. On the other hand, when Sarkosyl was added to 0.05% 30 sec before the addition of NTPs, transcript production was already maximal by 2 min after the start of transcription. This is evident from the constant signal in all lanes of Figure 6B. (Fig. 6A and B of this figure are from the same autoradiograph, thus allowing a direct visual comparison of the results of multiple-and singleround transcription. Later time points are not presented for the multiple-round transcription (Fig. 6A) because the autoradiograph is greatly overexposed.
We performed another experiment to determine how rapidly maximal transcript production was attained in the presence of 0.05% Sarkosyl, and the results are shown in Figure 6C. Full-length VAI RNA first appeared by 30 sec after the addition of NTPs, and its level was maximal by 60-75 sec. This time course clearly suggests that 0.05% Sarkosyl blocked reinitiation.
The presence of VA~ transcripts shorter than the fulllength product is also suggestive of limited reinitiation. These transcripts, which are clearly visible in the slightly overexposed autoradiograph shown in Figure  6C, most likely resulted from the release of RNA from ternary complexes containing polymerases paused at specific sites. Note that the amount of the longest putative pausing product is maximal at 45 sec of transcription, while a shorter one is maximal at 30 sec, suggesting that the shorter products are being chased into full-length transcripts. Sarkosyl has been shown to affect elongation by RNA polymerase II by inducing pausing downstream of the start site of a number of promoters, including adenovirus major late (Hawley and Roeder 1985) and HIV-1 and HIV-2 (Toohey and Jones 1989). Its effect on transcription elongation by RNA polymerase II appears to be much more stringent than its effect on RNA polymerase III, as the block to elongation on at least some fraction of the templates transcribed by RNA polymerase II could not be overcome by longer incubations (Hawley and Roeder 1985).
Sarkosyl at this higher concentration (0.05%) clearly limits reinitiation, and the most likely interpretation of these results is that it strictly blocks reinitiation by RNA polymerase III. To confirm this hypothesis, we performed the experiment diagrammed at the bottom of Figure 7. The template and factors were preincubated as in the experiments described above; however, in contrast to those experiments, transcription was initiated by the addition of NTPs in the absence of Sarkosyl. For

30' 30" At
Sarkosyl to NTPs .015% (A) or .05% (B and C) Figure 6. Addition of Sarkosyl to 0.05% after complex formation appears to allow only a single round of transcription, pVA~ and factors were preincubated for 30 min as described in the legend to Fig. 1. Sarkosyl was added to a final concentration of 0.015% (A) or 0.05% (B and C)just before the addition of NTPs. Transcription reactions were terminated by the addition of stop mix after the number of minutes (A and B) or seconds I C) shown above the lanes and were processed as described in Materials and methods. A and B are from the same autoradiograph, while C is the result of a separate experiment. Figure 7A, transcription was allowed to proceed for the amount of time shown above the lanes, at which point the stop mix was added. This shows the linear rate of VAI transcript production for 1 hr under these conditions. For Figure 7B, the times shown above the lanes represent the number of minutes between the addition of NTPs and the addition of Sarkosyl to a final concentration of 0.015%; transcription reactions were stopped 60 min after the addition of NTPs. Note that each of the signals in Figure 7B is equal in intensity to the signal resulting from 60 min of transcription in Figure 7A, directly supporting the claim made above that 0.015% Sarkosyl has no effect on reinitiation.
For Figure 7C, Sarkosyl was added instead to 0.05% at the same time points as for the addition to 0.015% in Figure 7B, again with 60 min between the addition of NTPs and the end of the transcription reaction with the addition of stop mix. The close correspondence between the signals in the corresponding lanes of Figure 7A and C indicates that even after transcription initiation and reinitiation, 0.05% Sarkosyl essentially acted as a stop mix by allowing elongation to proceed while strictly inhibiting reinitiation. The data in Figures 6 and 7 thus indicate that 0.05 % Sarkosyl can be used to assay single rounds of transcription.
If 0.05% Sarkosyl indeed allows only single rounds of transcription from all complexes, as opposed to multiple rounds from a Sarkosyl-resistant fraction of the total, then the initiation of transcription with a subset of the NTPs prior to Sarkosyl addition should result in a level of transcription comparable to that observed when Sarkosyl is added first. We performed such experiments in which an NTP mixture lacking ATP, CTP, or UTP was added to start production of a short transcript prior to the addition of Sarkosyl and, subsequently, the missing NTP. Levels of transcription were indeed approximately equivalent to those in which Sarkosyl was added before the NTPs (data not shown), strongly suggesting that single rounds of transcription are being assayed. We also performed experiments using heparin under conditions recently reported to limit yeast RNA polymerase III to single rounds of transcription (Kassavetis et al. 1989). The levels of transcription observed under these conditions were very similar to those seen with 0.05% Sarkosyl (data not shown). This provides even stronger evidence that our conditions allow only single rounds of transcription.
We have made an estimate of the number of active complexes in these assays. We determined that for the assays presented in Figure 6B, approximately 1 in 2000 of the VA~ templates present was transcribed. For the separate experiment presented in Figure 6C, roughly 1 in 5700 templates formed active complexes. Stated another way, in Figure 6B, 550 fmoles of pVAI produced 0.27 fmoles of VA~ RNA, and in Figure 6C conditions, active complexes support several hundred rounds of reinitiation per hour.

E1A expression results in an increased number of functional complexes
Our previous results (Hoeffler and Roeder 1985;Hoeffler et al. 1988) indicated that the stimulation of transcription by RNA polymerase III in extracts made from cells infected by adenovirus (and expressing E1A) was mediated by an increase in the level of a more active form of TFIIIC, while overall levels of TFIIIC, as measured by template commitment assays, were unchanged. Template commitment assays do not indicate, however, whether a stimulation of transcription results from an increase in the number of active transcription complexes or from an increase in the specific activity of a constant number of functional complexes {resulting, for example, from an increased frequency of polymerase reinitiation). To determine which of these possible mechanisms was responsible for the E1A-mediated increase in class III transcriptional activity, we used the ability of 0.05% Sarkosyl to block the reinitiation of transcription to assay single rounds of transcription in fractions derived from stimulated and unstimulated extracts. The experimental protocol is diagrammed in Figure 8. For this analysis, HeLa cells were first slowed in their growth by reducing the concentration of serum present in the medium (as detailed in Materials and methods). Then they were infected either with d1312, the adenovirus mutant lacking a functional E1A gene, or with d1309, the parent virus of d1312 having a wild-type E1A gene. Nuclear extracts were made at 6 hr postinfection and assayed for transcriptional activity. The dl309-infected cell extract had 7-to 10-fold higher activity than the control d1312-infected cell extract (data not shown). To deplete the extracts of contaminating nucleotides, which could complicate the analysis of single rounds of transcription, they were subjected to chromatography on parallel phosphocellulose columns. After washing to remove unbound nucleotides, TFIIIB, TFIIIC, and RNA polymerase III were eluted with a single step at 0.6 M KC1. As indicated in Figure 8, activity was assayed either by preincubation of the VAI template with d1309-or d1312-derived fractions prior to the addition of NTPs and a 1-hr transcription reaction (A) or by the addition of Sarkosyl to 0.05% just before the initiation of transcription in these fractions (B); as shown above, the latter conditions should limit transcription to a single round. The autoradioglaph of the resultant VAI transcripts are shown at the bottom of the figure. Figure 8A shows the level of stimulation of transcription after fractionation of the extracts. Equivalent volumes of fractions, which in this case correspond to cell equivalents, were assayed under conditions permitting multiple rounds of transcription. Essentially the same level of stimulation of transcription was also observed under single-round transcription conditions ( Figure 8B). The autoradiograph shown in Figure 8B was exposed much longer than the corresponding autoradiograph used for Figure 8A, to compensate for the greatly different intensities in signals (see figure legend for details); in any case, there was a clear difference between Figure 8. Enhanced VA I transcription in fractions derived from cells infected with adenovirus expressing E1A results from an increased number of functional preinitiation complexes. Cells were slowed in their growth and infected with d1309 or d1312 adenoviruses, and nuclear extracts were prepared as described in Materials and methods. Extracts were fractionated over phosphocellulose as described in Materials and methods, and the 0.6 M step fraction {containing TFIIIB, TFIIIC, and RNA polymerase III) was used in transcription assays as diagrammed. (A) 1.5, 2.5, 3.5, or 4.5 p,1 of a fraction derived from d1309-or dl312-infected cell extracts was preincubated with pVA~, MgC12, additional KC1, and H20 for 60 min prior to the addition of NTPs. Transcription reactions were terminated 60 rain later, and samples were processed as described in Materials and methods. The autoradiograph shown was exposed for 2.5 hr without an intensifying screen. (B) Preincubations and transcription reactions were exactly as described for A, except that Sarkosyl was added to a final concentration of 0.05% just before the addition of NTPs. The autoradiograph shown was exposed for 36 hr at -70°C with an intensifying screen. the levels of transcription in fractions derived from d1309-and d1312-infected cell extracts even when only a single round of transcription was permitted. This indicates that the stimulation of transcription resulted largely or entirely from an increase in the number of functional transcription complexes rather than from an increase in rates of reinitiation or elongation from an unchanged number of complexes.

Discussion
An understanding of the regulation of gene expression in eukaryotes requires detailed mechanistic information about the steps along the pathway of transcription complex formation and the initiation of transcription. The action of regulatory factors can be understood fully only by understanding which steps are rate limiting for transcription and how regulatory factors affect these steps. We used Sarkosyl to dissect functional steps in the assembly of a transcription complex on the VA~ gene, and we demonstrated the utility of this approach in determining the mechanism of the E1A-mediated stimulation of transcription of the VA~ gene.

Complexes resistant to Sarkosyl
Previous studies have indicated that the general order of association of proteins with the VAI promoter is TFIIIC, TFIIIB, RNA polymerase III, and that the resulting complexes are stable when challenged with competing templates (Lassar et al. 1983;Fuhrman et al. 1984;Bieker et al. 1985). The model shown in Figure 9 summarizes our present data about the effects of Sarkosyl on the func-tion of these complexes. As indicated, 0.015% Sarkosyl blocks the formation of a complex (complex I) whose function, once formed, is otherwise unaffected by this concentration of Sarkosyl. The simplest interpretation of our results (Figs. 4 and 5), showing that resistance of transcription to 0.015% Sarkosyl requires preincubation of the VA~ promoter with TFIIIC, TFIIIB, and RNA polymerase III, is that the resulting Sarkosyl-resistant complex involves stoichiometric interaction of all the factors. We have not excluded the possibility, however, that one of the components acts catalytically in forming a functional preinitiation complex that is resistant to Sarkosyl, nor do we know exactly which protein-DNA or protein-protein interaction is sensitive to Sarkosyl. RNA polymerase III itself also appears to be required for the formation of complex I (see Fig. 5), but may not be required for its maintenance. The latter is indicated by the ability of RNA polymerase III to exhibit multiple rounds of initiation, elongation, and termination in the presence of 0.015% Sarkosyl (present data) and by the corresponding inability of the polymerase to remain stably bound to a promoter complex containing TFIIIC and TFIIIB when challenged by a second equivalent complex formed in the absence of any RNA polymerase (Bieker et al. 1985). These latter studies, however, employed the 5S gene, as well as the regulatory factor TFIIIA, whereas gel filtration studies (Carey et al. 1986) have indicated that RNA polymerase III can be stably bound in a complex on the VA~ gene under conditions where it dissociates from a complex on the 5S gene. Thus, there are two likely explanations for the existence of multiple rounds of transcription on the VAI gene in the presence of 0.015% Sarkosyl: 1) the initial formation .015% .05%  of complex I, requiring RNA polymerase III, leads to a structural transformation in the VA~-TFIIIC-TFIIIB complex such that the Sarkosyl-sensitive step is not encountered during subsequent rounds of initiation, or 2) RNA polymerase III is actually a stably-bound component of complex I and does not dissociate completely from the VAI-TFIIIC-TFIIIB complex during transcription. Complex I exhibits a number of similarities to the previously defined rate-limiting complex on the 5S gene (Bieker et al. 1985). The kinetics of formation of these complexes are very similar, with a t~,2 of about 14 min, and all of the known protein components (TFIIIC, TFIIIB, RNA polymerase III, and, for the 5S gene, TFIIIA) are required in each case. Formation of the rate-limiting complex, however, also required the presence of a nucleotide (Bieker et al. 1985;Bieker and Roeder 1986), whereas the 0.015% Sarkosyl-resistant complex was formed in the absence of nucleotides. This difference could reflect dissimilarities in the assembly of complexes on the 5S and VA~ genes, or it may indicate that these steps, although apparently related kinetically, are distinct from each other.
The present studies have also defined a second complex (complex II in Fig. 9) whose formation is blocked by 0.05% Sarkosyl but which, once formed, can still initiate a single round of transcription at this concentration of detergent. This defines a step that, in contrast to the one inhibited by 0.015 % Sarkosyl, is encountered during each round of initiation and permits the assay of single rounds of transcription by RNA polymerase III. Nucleoside triphosphates are also not required for the formation of complex II; this property distinguishes complex II from the elongation complex containing template, elongating RNA polymerase, and nascent RNA. The relationship of complexes I and II is still unclear; they may differ as a result of a structural transition that is analogous, for instance, to the closed-to-open promoter conversion in prokaryotic systems (for review, see von Hippel et al. 1984), or they may differ by the presence of an additional cofactor. In this regard, it has been reported that TFIUC can be split into two components, TFIIIC1 and TFIIIC2 (Dean and Berk 1987;Yoshinaga et al. 1987), the latter being involved in the formation of the initial stable complex. We have not been able to investigate the role of TFIIIC 1 or possible alternative complex formation pathways at this time because we have been unable to detect separable activities (R. Kovelman and R.G. Roeder, unpubl.}, even with extensive purification of TFIIIC (Cromlish and Roeder 1989).

Comparisons between RNA polymerase III-and RNA polymerase II-dependent promoters
There are both similarities and differences between the effects of Sarkosyl on the VAI gene {this report) and on the adenovirus major late (ML) gene [Hawley and Roeder 1985). Each transcription system shows two distinct steps sensitive to different concentrations of Sarkosyl, and in each case the data are consistent with transcription being limited to a single round under certain conditions. The specific effects of particular Sarkosyl concentrations, however, differed for the VAx and ML promoters. Thus, although the addition of 0.015% Sarkosyl at the beginning of the preincubation completely blocked transcription in each system, addition after the preincubation restricted transcription by RNA polymerase II to a single round but was without any detectable effect on reinitiation by RNA polymerase III. In other words, a step sensitive to disruption by 0.015% Sarkosyl is encountered during each round of transcription initiation by RNA polymerase II but is encountered only once [i.e., at the formation of complex I (Fig. 91] during transcription by RNA polymerase III. This difference may reflect the distinct characteristics of stable complexes on class II versus class III genes; thus, all of the accessory factors required for initiation by RNA polymerase III can be sequestered into a stable promoter complex (Lassar et al. 1983;Bieker et al. 19851, whereas some of the accessory factors required by RNA polymerase II cannot be IVan Dyke et al. 1989). Another difference is that a higher concentration of Sarkosyl {0.05%1 was required to block the reinitiation of transcription by RNA polymerase III. Moreover, and significantly, nucleotides were not required to form Sarkosylresistant complexes capable of supporting single rounds of transcription by RNA polymerase III, whereas the apparently analogous conversion of the class II rapid start complex to a Sarkosyl-resistant complex required the presence of NTPs (Hawley and Roeder 1985). This difference in nucleotide requirements may correspond to the demonstrated need for a hydrolyzable 13-~/bond in ATP or an ATP analog for transcription in the class II system (Bunick et al. 1982;Sawadogo and Roeder 19841 but not in the class III system (Bunick et al. 1982;Carey et al. 1986).

Mechanisms of stimulation of transcription
The adenovirus E 1A protein stimulates the transcription of a number of viral and cellular genes (for review, see Berk 1986), and these effects appear to be mediated via a number of different cellular transcription factors. For example, the cellular factors E2F and E4F have been implicated as targets for E1A activation of the adenovirus E2A and E4 genes, respectively (Kovesdi et al. 1986a, b;Lee and Green 1987;Raychaudhuri et al. 1987;Hardy et al. 1989), whereas mutagenesis has demonstrated the importance of the TATA element, and thus a general transcription factor that interacts directly (TFIID) or indirectly (e.g., TFIIB, TFIIE, or RNA polymerase II) with this element, in E1A action on the adenovirus E1B (Wu et al. 1987) and human hsp70 (Simon et al. 1988) genes.
TFIIIC was shown to be another target for E1A activation by the apparent induction of a distinct form of the factor with higher transcriptional activity on adenovirus infection (Hoeffler et al. 1988). For a number of these cases, phosphorylation has been implicated in the modulation of transcriptional activity (Hoeffler et al. 1988;Bagchi et al. 1989;Raychaudhuri et al. 1989}, while in other cases, transcriptional activation by E 1A appears to require the activity of a viral early protein from the E4 coding region (Babiss 1989;Hardy et al. 1989;Huang and Hearing 1989;Reichel et al. 1989). In spite of the increased understanding of some of the direct and indirect effects of E1A, the biochemical mechanisms by which levels of transcription are increased--in particular, the steps most closely associated with the initiation of transcription at the target gene--are still unknown. In fact, the stimulation of RNA polymerase II transcription by upstream promoter-binding factors, some of which have been extensively characterized with respect to activation domains (for review, see Maniatis et al. 1987;Ptashne 1988), is also not understood mechanistically. In at least some cases, quantitative and/or qualitative effects of the regulatory proteins on the basal transcription complex have been observed (Horikoshi et al. 1988a, b;Workman et al. 1988).
Previous results of in vitro transcription on fractionated extracts (Hoeffler and Roeder 1985;Yoshinaga et al. 1986) and of gel retardation assays (Hoeffler et al. 1988) have clearly implicated TFIIIC in E1A-mediated enhancement of VAI transcription. It was reported on the basis of template commitment assays that adenovirus infection led to a quantitative increase in the total amount of TFIIIC that could bind to the VA~ gene (Yoshinaga et al. 1986), but our data indicated that there was no such change in total factor levels (Hoeffler et al. 1988). Taking advantage of our ability to measure single rounds of transcription, we demonstrate here (Fig. 8) that this stimulation of transcription results from an increased number of transcription complexes. It may appear that this result contradicts our previous finding, which was based on template commitment assays, that overall levels of TFIIIC did not change; however, the Sarkosyl inhibition of RNA polymerase II template commitment assay is a DNA binding assay and does not distinguish between active and inactive complexes. We also showed, by gel retardation assays, that E1A expression resulted in a marked increase in the proportion of TFIIIC that was transcriptionally active {Hoeffler Kovelman et al. 1989; R. Kovelman and R.G. Roeder, unpubl.). Therefore, template commitment assays, gel retardation assays, and single-round transcription assays indicate, altogether, that the E1Amediated enhancement of VAI transcription results from an increased number of preinitiation complexes containing a transcriptionally active form of TFIIIC. Mechanistically, this is currently the best understood example of E1A action at the promoter level, whether this action is direct or indirect. Even if the stimulation of transcription by E1A operates via more than one pathway, this system is likely to provide information toward an understanding of other mechanisms.
Stimulation of VA~ transcription in vitro that results from an increased number of active complexes could also be of direct relevance in vivo during the course of adenovirus infection. The levels of VA~ RNA increase dramatically only after viral replication {S6derlund et al. 1976); this may be caused not only by an increased number of templates, but also by a greater proportion of those templates being in an active state. Immediately after replication, VA~ templates may form either active or inactive transcription complexes, and the relative proportions of the different forms of the key initiation factor TFIIIC may determine overall levels of VA I transcription. It has been demonstrated under various in vitro conditions that TFIIIA or TFIID can prevent the nucleosome-mediated repression of transcription (Bogenhagen et al. 1982;Gottesfeld and Bloomer 1982;Workman and Roeder 1987), and that the pseudorabies virus immediate early protein, which can stimulate the transcription of many of the same genes as the adenovirus E1A protein, further potentiates the formation of active transcription complexes on the adenovirus ML promoter under nucleosome assembly conditions (Workman et al. 1988 I. The regulation of transcriptional levels via competition between the formation of active and inactive complexes may be a significant mode of regulation of all multicopy genes and may also be relevant for the regulation of some single-copy genes. Sarkosyl and other inhibitors of particular steps of transcription will continue to be useful in helping to delineate the steps themselves as well as to determine how various regulatory proteins affect the key rate-limiting steps.

Cells and viruses
Maintenance of HeLa cells (obtained from G. Attardi) in standard Joklik minimal essential medium (MEM) containing 5.0% bovine calf serum or low-serum (0.5% BCS) Joklik-MEM was as described previously (Hoeffler et al. 1988).
Adenovirus type 5 mutants d1309 (obtained from L. Babiss) and d1312 (obtained from H. Ginsberg) were titrated on HeLa or 293 cell monolayers, respectively, by plaque assay (Williams 1970). Except for the use of AdS d1309 instead of wild-type Ad2, infections of HeLa cells grown in 0.5% serum-containing medium were as described previously (Hoeffler et al. 1988).

Extracts and fractionation
Nuclear extracts were prepared from uninfected HeLa cells essentially as described elsewhere (Dignam et al. 1983), whereas nuclear extracts from virus infected cells were prepared according to the slightly modified method of Hoeffler et al. (1988).
Phosphocellulose fractionation of uninfected cell nuclear extracts on phosphocellulose (Whatman P11) was performed essentially as described previously for S100 extracts {Segall et al. 1980). Extracts were loaded onto columns equilibrated in buffer A [20 mM HEPES-NaOH (pH 7.9) or 20 mM Tris-HC1 (pH 7.9), 20% (vol/vol) glycerol, 0.2 mM Na~ EDTA, and 1 mM dithiothreitol] containing 0.1 M KC1. The column was washed with this buffer, and proteins were sequentially step-eluted with buffer A containing 0.35 M KC1 and 0.6 M KC1. Protein concentrations were determined according to the method of Bradford (1976). Fractions containing peak concentrations of protein were pooled, dialyzed against buffer A plus 0.1 M KCI for 2-3 hours, and stored at -70°C. Most of the RNA polymerase III activity was in the 0.35 M KCI step, but approximately 10% was in the 0.6 M KC1 step. These fractions were used for all experiments except those for Figures 5 and 8.
Fractionation of infected-cell nuclear extracts (analyzed in Fig. 8) was performed essentially as described in the preceding paragraph, except that the 0.35 M KC1 step was omitted. The single step at 0.6 M KC1 therefore contained all of the proteins required for VA~ transcription.
For the RNA polymerase III used in the experiment of Figure  5, 1 liter of HeLa cell S100 extract (prepared as described in Weil et al. 1979) was loaded onto a 300-ml phosphocellulose column equilibrated in buffer A plus 0.1 M KC1. Proteins were eluted with a 4 column-volume gradient from 0.1 M KC1 to 1.0 M KC1 in buffer A. Fractions containing the peak of polymerase activity were pooled and dialyzed against buffer B containing 50 mM (NH4)2SO 4. Successive chromatography on DEAE-cellulose (Whatman DE52) and DEAE-Sephadex A-25 {Pharmacia), followed by step-elution from phosphocellulose to concentrate the sample eluted from Sephadex A-25, was performed essentially as described in Sklar and Roeder (1976), except that the final phosphocellulose column was run in KC1 instead of (NH4)2SO 4. The fractions assayed in the experiment shown in Figure 5 were >95% free of cross-contamination.

Transcription assays
Standard 25-~1 transcription reactions contained 70 mM KC1, 7 m~ MgClz, 8% (vol/vol) glycerol, 8 mM HEPES-NaOH (pH 7.9), 0.4 mM DTT, 5 mM creatine phosphate, 40 p~g/ml pVA~, 600 ~M each ATP, CTP, and UTP, and 12.5 ~M [ot-a2P]GTP (8.0 Ci/ mmole). Unlabeled nucleoside triphosphates were obtained from Pharmacia; [cx-a2P]GTP (3000 Ci/mmole) was obtained from Amersham. Sarkosyl (N-lauroylsarcosine, sodium salt) was obtained from Sigma. Sarkosyl solutions were made up as a 20% (wt/vol) stock. [We noticed some variation in the concentration dependence of the Sarkosyl effects reported with the batch of Sarkosyl. A new solution must be titrated to determine the optimal concentrations for assaying complexes I or II (see Fig. 9)]. All concentrations of standard transcription reaction components as well as of Sarkosyl are expressed as final concentrations during the transcription reaction itself. Because all components except for the NTPs, Sarkosyl (when added after the preincubation), and some protein fractions (Fig. 4) were incubated from the beginning of the preincubation, the actual concentrations of KC1, MgC1 v etc., were somewhat higher during the preincubation than stated above and in the figure legends.
Transcription reactions were stopped by the addition of 25 ~1 of a mixture containing 100 mM sodium acetate (pH 5.5), 0.4% SDS, 2 mg/ml yeast RNA, and 30 mM Na 2 EDTA, followed by sequential extractions with 2.5 volumes of phenol and chloroform. RNA was precipitated by the addition of 2.5 volumes of a 90% ethanol/100 mM sodium acetate (pH 5.5) mixture. Pellets were dried and resuspended in 5 ~1 H20. Formamide (10 ~1), containing 0.1% bromophenol blue and 0.1% xylene cyanole, was added, and the samples were incubated for 5 min at 95°C prior to being loaded onto an 8% polyacrylamide/7 M urea/1 x TBE (89 mM Tris, 89 mM boric acid, 2.5 mM Na 2 EDTA) gel that had been prerun for 30 min at 200 V. Samples were electrophoresed for approximately 2.5 hr at 200 V, and the gel was soaked in H20 for 15-20 min and dried prior to autoradiography.

Quantitation
Incorporation of radioactivity into VAt RNA was quantitated by the excision of the relevant slices of the dried gels followed by counting in scintillation fluid (Liquiscint; National Diagnostics). Slices of equal area from the region of the gels immediately above and below the VA I transcript were also excised, and the averages of the counts of these slices were subtracted from the specific transcript incorporation to correct for background radioactivity. This background was a very small number compared to the incorporation of a2p into VA~ RNA under multipleround transcription conditions, but it typically represented 20-50% of the apparent radioactivity incorporated under single-round conditions. No attempt was made to correct for the quenching effect of polyacrylamide; moreover, as noted in Results, we noticed substantial variation in the amount of radioactivity incorporated with the age of the [c~-a2P]GTP. Therefore, our estimates of the number of active complexes should be considered approximate. Nonetheless, direct relative comparisons (as in Fig. 8) can be made.