©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nucleotide Requirements for Activated RNA Polymerase II Open Complex Formation in Vitro(*)

(Received for publication, July 8, 1994; and in revised form, October 12, 1994)

Ying Jiang Jay D. Gralla (§)

From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90024-1569

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The role of nucleotides in activated RNA polymerase II transcription was studied. Permanganate footprinting confirmed that there is a strict nucleotide requirement for forming open promoter complexes that cannot be overcome by the addition of a dinucleotide primer corresponding to the start site sequence. However, higher concentrations of other nucleoside triphosphates can substitute for ATP in catalyzing open complex formation. Opening catalyzed by these nucleotides is inhibited by the ATP analogue adenosine 5`-O-(thiotriphosphate), suggesting that they may function through cross-binding to the ATP site. The K for ATP for opening and the involvement of other nucleotides in opening differs from the characteristics reported for TFIIH helicase and C-terminal domain kinase activities. This raises the possibility that opening does not involve these activities. The results alleviate very significantly the considerable current uncertainty concerning the role of ATP in the mammalian mRNA transcription initiation pathway.


INTRODUCTION

Transcription initiation by RNA polymerase II proceeds through a multistep pathway. First, protein factors interact with DNA elements to form a closed promoter complex. Then, the closed complex isomerizes into an open complex in which the DNA template strand is exposed for transcription. This is followed by the initiation of RNA synthesis. Sometime after RNA chain initiation has begun, RNA polymerase leaves the promoter and progresses into the transcription elongation mode (sometimes termed promoter clearance). This pathway involves a common set of general transcription factors (activator, TFIID, A, B, pol II, (^1)F, E, H and J), but the individual roles of most of these factors remain unsettled. The factors can be assembled onto the DNA in an ordered or stepwise pathway (for review, see Conaway and Conaway(1993) and Zawel and Reinberg(1993)), or alternatively, pre-assembled multifactor complexes can be recruited onto the promoter (Koleske and Young, 1994; Kim et al., 1994). In the presence of nucleoside triphosphates these assemblies catalyze the transcription initiation cycle.

Many studies have concluded that hydrolysis of the beta- bond of ATP is required for pol II transcription initiation (Bunick et al., 1982; Sawadogo and Roeder, 1984; Conaway and Conaway, 1988; Jiang et al., 1993). However, there is considerable uncertainty about what steps involve ATP hydrolysis. A number of steps have been proposed as requiring ATP, including open complex formation (Wang et al., 1992a, 1992b; Jiang et al., 1993) and promoter clearance (Goodrich and Tjian 1994). ATP is also required for helicase and kinase activities possessed by factors associated with transcription initiation (Feaver et al., 1991; Lu et al., 1992; Serizawa et al., 1992, 1993; Schaeffer et al., 1993). The relationships between these various ATP-requiring processes and those ATP-dependent enzymatic activities are not known but have been the subject of much discussion.

The open complex step has been studied quite intensively and has been found to require ATP at a variety of promoters that depend on transcription activators (Wang et al., 1992a, 1992b; Jiang et al., 1993). Recent transcription studies have raised the possibility that in certain systems ATP may not be required to open the transcription start site (Goodrich and Tjian 1994; Timmers 1994). In an attempt to reconcile these various experiments, we have adopted selected conditions used in the minimal systems and directly measured pol II open complex formation. The data show that promoter opening has a strict nucleotide requirement but that high concentrations of other nucleotides can substitute for ATP, possibly by cross-binding. Other results argue against, but do not exclude, that the use of ATP for opening is via the activity of the TFIIH helicase or of the C-terminal domain (CTD) kinase.


MATERIALS AND METHODS

High pressure liquid chromatography-purified ribonucleoside triphosphates and deoxyribonucleoside triphosphates were purchased from Pharmacia Biotech Inc. Elution profiles, provided by the manufacturer, demonstrate that the nucleotides are 99% pure and show no cross-contamination of other nucleotides with ATP. Three different batches of nucleotides gave similar results. alpha-Amanitin was from Sigma and was present at a concentration of 1 µg/ml. Hela cell nuclear extract was prepared as described (Dignam et al., 1983). Several batches of nuclear extract were used in these experiments. All showed an absolute requirement for nucleotide in the opening reaction, with ATP acting at the lowest concentration. The absolute values of the K values, however, varied somewhat with the extract preparation.

The DNA template is as described (Jiang and Gralla, 1993; Wang et al., 1992a; Carey et al., 1990a, 1990b) and is a supercoiled plasmid containing nine GAL4 binding sites upstream of truncated Adenovirus E4 promoter, which retains its core promoter element and transcription start site region. In some experiments, plasmid template was linearized by BglI digestion, as indicated.

Potassium permanganate footprinting was as described (Wang et al., 1992a, 1990b; Jiang et al., 1993, 1994; Jiang and Gralla, 1993). First, the transcription mixture was set up which contains 25 µl of Hela nuclear extract (6 mg/ml protein concentration in D buffer: 20 mM Hepes, pH 7.9, 100 mM KCI, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), 8.25 mM magnesium chloride, 200 ng of carrier plasmid DNA, 10 ng of supercoiled DNA template or 12 ng of linearized DNA template. GAL4-VP16 or GAL4-AH was added as transcription activator. This mixture was incubated for 30 min to allow the assembly of preinitiation complexes. Then dATP or ATP was added for 2 min to drive promoter opening. Potassium permanganate was added at 6 mM for 3 min to probe the single strand region of DNA. 2-Mercaptoethanol was used to quench the potassium permanganate. Proteinase K was added to digest proteins, which was followed by phenol, phenol/chloroform, chloroform extraction. DNA was precipitated and redissolved in water and was passed through a Sephadex G50-80 spin column to desalt. Taq polymerase was used to extend a radiolabeled primer repetitively, and the products were resolved on a 6% sequencing gel. Potassium permanganate hypersensitive sites, indicative of DNA melting, were revealed on the autoradiograph.

The investigation of Mg and nucleotide requirements for open promoter complex formation basically was modeled on the above procedure. Mg was omitted from the transcription mixture to study the Mg requirement. Other nucleotides or analogs were added at concentrations as indicated.


RESULTS

Transcription Start Site Opening Is Driven by ATP beta- Bond Hydrolysis and Requires Mg

ATP is required for open promoter complex formation in response to transcription activators (Wang et al., 1992a, 1992b; Jiang et al., 1993, 1994; Jiang and Gralla, 1993). The amount of ATP required has not been determined. In this set of experiments, we determine the amount of ATP required to open the transcription start site and also establish a divalent magnesium ion requirement. The system uses the G9E4T DNA template, which has nine GAL4 binding sites upstream of a truncated form of the adenovirus E4 promoter. The transcription activator is GAL4-AH. Hela nuclear extract was used as a source of general transcription factors. In this system, strong permanganate reactivity appears over the transcription start site when a functional transcription complex forms (Wang et al., 1992a, 1992b; Jiang et al., 1993, 1994; Jiang and Gralla, 1993). This is a consequence of permanganate reacting preferentially with single-stranded DNA. This is detected as a hyperreactive region on a DNA sequencing gel using footprinting-type protocols.

Lanes1 and 2 of Fig. 1are repeats of prior experiments and confirm that opening does not occur in the absence of ATP or in the absence of activator (compare the absence of signals to the positive control in lane6). Lane3 shows that opening also does not occur in the absence of divalent magnesium ion. The figure also shows that opening occurs in the presence of ATP (lane6) but does not occur using the nonhydrolyzable ATP analogs AMP-PNP (lane4) or ATPS (lane5). Therefore, in agreement with prior studies, pol II open complex formation requires ATP beta- phosphoanhydride bond hydrolysis. The experiment also shows that opening requires magnesium ion, which is an established co-factor in ATP-dependent reactions.


Figure 1: Pol II open promoter complex formation requires hydrolyzable ATP and Mg. 10 ng of supercoiled G9E4T DNA template were incubated with HeLa nuclear extract, GAL4-AH, and 8.25 mM Mg for 30 min to allow the assembly of preinitiation complexes. Then, dATP was added at 500 µM for 2 min to drive promoter opening (lane6), followed by potassium permanganate probing. Comparisons include: lane1, without any nucleotide; lane2, without activator; lane3, without Mg; lane4, 1 mM AMP-PNP was added instead of dATP; lane5; 500 µM ATPS was added instead of dATP. The start site region is bracketed and the -10 position permanganate sensitivity is indicated. Lanes1 and 2, 3 and 4, and 5 and 6 were from three separate experiments.



Next we determined whether opening could also occur at very low concentrations of ATP. Fig. 2shows a titration using various concentrations of ATP or dATP and either of 2 activators: GAL4-AH and ATP (Fig. 2A), GAL4-VP16 and dATP (Fig. 2B). In both cases, the opening reaction is not detectable at 2.5 µM ATP or dATP (Fig. 2, A and B, lanes3) and is quite strong at 10 µM ATP or dATP (Fig. 2, A and B, lanes1). Other experiments (not shown, but see below) indicate that the reaction is not quite complete at 10 µM and suggest a K(M) of 5-10 µM for ATP or dATP. Because the opening reaction is shown to have an absolute requirement for added low concentrations of ATP, the endogenous amounts of ATP must be extremely low or absent.


Figure 2: Titration of ATP in pol II open complex formation. Preinitiation complexes were assembled for 30 min and then various concentrations of ATP or dATP were added for 2 min before potassium permanganate probing. A, GAL4-AH was added as transcription activator. Lane1, 10 µM ATP; lane2, and 5 µM ATP; lane3, 2.5 µM ATP; lane4, 1.25 µM ATP; lane5, without ATP. B, GAL4-VP16 was the transcription activator, and dATP was used instead of ATP, otherwise lanes are as in A. The start site region is bracketed, and the -10 position permanganate sensitivity is indicated.



Higher Concentrations of UTP, CTP, and GTP, But Not a Dinucleotide Primer, Can Substitute for ATP in Driving pol II Open Complex Formation

Recent experiments in a minimal basal transcription system showed that abortive RNA transcripts could be produced invitro in the presence of a dinucleotide primer and the next templated nucleoside triphosphate (Goodrich and Tjian, 1994). Because this reaction lacked ATP, the possibility was raised that the ATP requirement might be imposed by the use of an unfractionated transcription system. In the next set of experiments we use the unfractionated system and explore alternative possibilities for how opening might occur under these abortive conditions.

One possibility is that opening can occur due to the presence of a dinucleotide primer; this could conceivably trigger opening by binding to the polymerase-promoter complex and forcing the strands apart as it hybridized to the template. To test this possibility we used UpU, which is complementary to the transcription start site of the adeno E4 promoter used here. Lanes2 and 3 of Fig. 3show that even 2 mM UpU dinucleotide fails to trigger opening (compare with lane1 in which opening is driven by dATP). Thus a complementary dinucleotide primer cannot substitute for ATP in driving promoter melting even at a concentration that is approximately 200 times greater than the K(M) for ATP.


Figure 3: UTP but not UMP or dinucleotide UpU can substitute for ATP to drive promoter opening. Closed preinitiation complexes were assembled for 30 min with or without alpha-amanitin, and then different nucleotides were added before potassium permanganate probing. Lane1, 500 µM dATP was added for 2 min; lane2, 2 mM UpU was added for 2 min; lane3, 2 mM UpU was added for 8 min; lane4, 500 µM ATP was added for 2 min; lane5, 2 mM UpU and 125 µM UTP were added for 2 min; lane6, 125 µM UTP was added for 2 min; lane7, alpha-amanitin was added before 2 min of 125 µM UTP addition; lane8, 1 mM UMP was added for 2 min. The start site region is bracketed, and the -10 position permanganate sensitivity is indicated. In the absence of alpha-amanitin, when UpU together with UTP or UTP alone used to drive promoter opening, the potassium permanganate hypersites reach +18 position as indicated. Lanes1-3, 4 and 5, 6 and 7, and lane8 were from separate experiments.



Next we added 125 µM UTP to the reaction containing UpU primer to simulate conditions that would allow RNA chain initiation to begin. Transcripts made in the presence of UpU and UTP can begin with the sequence UpUpU. Permanganate probing shows that a series of changes is induced by the inclusion of UTP in a UpU-primed reaction (lane3 with UpU compared with lane5 with UpU + UTP). Very strong opening is indicated by the strong permanganate signal in lane5, which includes the usual start site region (indicated by brackets). The signal includes additional bands downstream, within the initial transcribed region, up to +18. However, comparison experiments show that this opening reaction does not depend on the presence of dinucleotide UpU; lane6 of Fig. 3shows that both start site and downstream opening can be triggered by 125 µM UTP in the absence of UpU.

This result suggests that UTP can trigger opening by virtue of it being a hydrolyzable substrate as was the case with ATP. This is supported by other experiments. Neither UpU or UMP trigger the opening reaction (UpU, lanes2 and 3; UMP, lane8). In addition, alpha-amanitin does not inhibit the UTP-dependent opening over the start site (lane7), showing that initial RNA synthesis is not required under these conditions. alpha-Amanitin does change the pattern, however, in restricting the signal to the start site as opposed to the initial transcribed region (compare lane7 with lane6). This suggests that the part of the signal that lies within the initial transcribed region corresponds to transcription bubbles associated with the beginning of RNA synthesis (further discussed below). However, the part of the signal that corresponds to the typical open complex over the start site does not disappear, confirming that UTP triggers opening independent of RNA synthesis.

Because the opening triggered by UTP is not dependent on the formation of a transcript, it is possible that other nucleotides that do not correspond to the initial transcript could also trigger opening. The data in Fig. 4show that as ATP, 125 µM of either GTP, CTP, or UTP, can also drive promoter opening (lanes 2-5versuslane1 without nucleotide added). Because alpha-amanitin was added to these reactions before nucleotides were added, the downstream hyper-sites triggered by UTP, which reflect RNA chain initiation, are not observed (lane3). The data show that there is a strict nucleotide requirement for opening but that this can be satisfied by high amounts of other nucleoside triphosphates.


Figure 4: All four NTPs can be used for promoter opening and ATPS inhibits open complex formation driven by UTP, CTP, and GTP. Closed preinitiation complexes were assembled for 30 min and then alpha-amanitin was added before a 2-min nucleotide addition and probing. Lane1, without any nucleotide; lane2, 125 µM ATP; lanes3 and 7, 125 µM UTP; lanes4 and 9, 125 µM CTP; lanes5 and 11, 125 µM GTP; lane6, 125 µM ATPS was added before 125 µM UTP addition; lane8, 125 µM ATPS was added before 125 µM CTP addition; lane10, 125 µM ATPS was added before 125 µM GTP addition. The start site region is bracketed, and the -10 position permanganate sensitivity is indicated.



These same results were obtained with several different batches of nucleotides purified by fast protein liquid chromatography. Elution profiles showed no cross-contamination with ATP and a purity greater than 99%. Since no signal is seen even when 2.5 µM ATP is added (above) neither cross-contamination nor endogenous ATP can account for the opening seen when other nucleotides are added.

In an attempt to determine whether these nucleotides use the same enzyme site as does ATP, we added ATPS to each reaction before UTP, CTP or GTP was added (lanes6, 8, and 10). Recall that ATPS does not have a beta- phosphoanhydride bond that is easily hydrolyzed and thus does not trigger opening (Fig. 1, lane5). This property has made ATPS a useful competitive inhibitor of many enzyme reactions that rely on the binding and hydrolysis of ATP. The results show that the 125 µM ATPS inhibits opening driven by equal amounts of UTP, GTP, or CTP (Fig. 4, lanes6, 8, and 10versuslane 7, 9, and 11). This suggests that all nucleotides use a similar mechanism to open the start site and that in fact they may all cross-bind to the same enzyme site, where ATPS also binds.

ATP Is Preferred in Driving Pol II Promoter Melting

In order to determine if there is a preference for the use of ATP, the assay was repeated at 10 µM, which is slightly higher than the K(M) for ATP determined above. The results (Fig. 5A) show that ATP (lane1) is clearly the preferred nucleotide with CTP (lane3) and UTP (lane4) yielding significantly lower signals and GTP yielding a barely detectable signal (lane2). Thus ATP is the preferred substrate, but other nucleoside triphosphates can substitute for ATP at higher concentration, probably by cross-binding.


Figure 5: ATP is the preferred substrate for pol II open complex formation. A, DNA template (10 ng) was added as supercoiled plasmid. In the presence of alpha-amanitin, open complexes were assayed in a standard protocol using 10 µM nucleotide; ATP (lane1), GTP (lane2), CTP (lane3), and UTP (lane4). B, the DNA template (12 ng) was linearized prior to addition to the HeLa extract; 10 µM ATP (lane1), GTP (lane2), CTP (lane3), and UTP (lane4). The start site region is bracketed, and the -10 position permanganate sensitivity is indicated.



Recent experiments have shown that the requirements for transcription can differ depending on whether linear or supercoiled DNA is used (Parvin and Sharp, 1993; Timmers, 1994; Goodrich and Tjian, 1994). In addition, it is well established that the open complex formation by prokaryotic polymerases can be facilitated by DNA supercoiling (Meiklejohn and Gralla(1989) and for review, see Pruss and Drlica(1989)). In these and in all of our previous experiments, the template was added to reactions in the supercoiled form. The state of the template during the subsequent incubation has been followed and it has been found that it is gradually converted to forms with lower superhelical density and to concatamers. (^2)To investigate whether these results are influenced by changes in DNA topology, the experiments were repeated using DNA that had been linearized prior to incubation. The result shows that the preference of ATP is retained using a linear DNA template (Fig. 5B).

The Melted DNA Bubble Does Not Appear to Change in Size during Initiation

The above experiments also suggest the possible size of melted DNA bubble of the pol II transcription complex. When dATP or ATP is used to trigger promoter opening in the presence or absence of alpha-amanitin, the potassium permanganate hypersensitive sites have their furthest extents at positions -10 and +4 (Fig. 1, lane6; Fig. 2; Fig. 3, lane1 and 4; Fig. 4, lane2; Fig. 5A, lane1; Fig. 5B, lane1). This is also true when GTP or CTP is added to trigger promoter opening (Fig. 4, lanes4, 5, 9, and 11). When UTP was used in the presence of alpha-amanitin, which prevents RNA synthesis, the potassium permanganate pattern was similar to dATP alone (Fig. 3, lane7; Fig. 4, lanes3 and 7). These data suggest that the transcription bubble lies between -10 and +4 prior to transcription initiation and thus is approximately 14 base pairs long.

These open complex patterns of melting can be compared with those seen when initial transcription is allowed. When UTP is used in the absence of alpha-amanitin, short transcripts ranging from 2 to 6 nucleotides can theoretically be produced, corresponding to initiation at any of 6 adjacent thymines of the E4 promoter transcription start site. The data shown that the use of UTP leads to changes in the pattern of the permanganate sensitivity; the strongest region of sensitivity now extends from -3 and +18 (Fig. 3, lanes5 and 6). This 21-base pair region is expected to include complexes associated with transcription of the 6 thymines in the start site region. Thus it likely represents a mixture of up to 6 overlapping transcription bubbles. These bubbles would each need to be 15 base pairs long to occupy cumulatively a 21-base pair region. This suggests that the bubble maintains a constant length of 14-15 base pairs during the transition from open complex to initial transcribing complex. The results suggest similarities with prokaryotic transcription, which has a similar bubble size (Siebenlist etal., 1980; Kirkegaard etal., 1983; Carpousis and Gralla, 1985; Sasse-Dwight and Gralla, 1989) which remains constant during transcription (Gamper and Hearst, 1982).


DISCUSSION

It has been known for many years that the elongation substrate AMP-PNP, an analog of ATP that contains a nonhydrolyzable beta- bond, cannot substitute for ATP in pol II transcription (Bunick et al., 1982). Inclusion of dATP in a AMP-PNP reaction restores accurate pol II transcription (Sawadogo and Roeder, 1984; Conaway and Conaway, 1988; Jiang et al., 1993). These observations demonstrate the involvement of ATP and the hydrolysis of its beta- bond in transcription initiation by RNA polymerase II. However, recent studies involving minimal transcription systems have found conditions where ATP does not appear to be required for initiation (Goodrich and Tjian 1994; Timmers 1994).

It was shown previously that formation of a pol II open promoter complex has the same ATP requirements as does transcription (Wang et al., 1992a; Jiang et al., 1993). In this paper we confirm that there is a strict nucleotide requirement for open complex formation. However, we also find that other nucleotides can substitute for ATP when they are present at higher concentrations. Because ATP is the preferred substrate and because the intracellular concentration of ATP is by far the highest of the nucleotides, we infer that promoter opening in vivo is driven primarily by ATP. However, in vitro, whether the promoter opens will be determined by the aggregate effects of all nucleotides that are present.

These observations may be used to rationalize some apparently conflicting observations in the literature. Recent studies have shown that transcription initiation can be detected in the absence of ATP. One case used an abortive initiation system (Goodrich and Tjian, 1994). Such systems typically involve reiterative synthesis of short RNAs, which can be 100 times more abundant than long transcript. Thus even a small fraction of DNA in a functional state could yield a detectable signal in this assay under conditions where opening and long transcripts would be much more difficult to detect. All systems (Timmers, 1994; Parvin and Sharp, 1993; Goodrich and Tjian, 1994) use fully supercoiled DNA, which allows the apparent bypass of several factor requirements, including that for hydrolyzable ATP and activator. All systems contain other hydrolyzable nucleotides, which the current results imply may partially substitute for ATP in triggering opening. This suggests that the lack of requirement for ATP in abortive initiation was related to an ability to detect minor fractions of transcription complexes formed in the presence of suboptimal amounts of substitute nucleotides.

ATP has also been suggested to be required for other steps in the transcription pathway, including promoter clearance (Goodrich and Tjian, 1994). Our results do not address this issue directly, but they support this possibility. The proposed ATP requirement for promoter clearance has two properties that appear to differ significantly from the ATP requirement for opening. The clearance step apparently cannot be facilitated by other nucleotides, whereas other nucleotides can facilitate the opening step. In addition, the ATP requirement for clearance can be bypassed by DNA supercoiling, whereas in the Hela extract the ATP requirement for opening cannot be bypassed by DNA supercoiling; however, this would need to be verified in a more purified system. Nonetheless, it seems likely that polymerase II transcription initiation involves two successive ATP-using steps that are catalyzed by different enzyme activities.

There has been much discussion of the role of the ATP-dependent DNA helicase and CTD kinase activities in transcription (Corden, 1990; Young, 1991; Guzder et al., 1994; Jiang and Gralla, 1994). Our results raise the possibility that these activities might not be involved in promoter opening. Recall that the data show that the K(M) for ATP is 5-10 µM for opening and that other NTPs can partially substitute for ATP. By contrast, the helicase activity has a K(M) for ATP of nearly 200 µM and can use only ATP. The CTD kinase activity has a different nucleotide requirement than that for opening. It works with ATP and GTP, with ATP being preferred; which differs from the promoter opening activity. These considerations suggest that promoter opening occurs independent of the DNA helicase and CTD kinase activities. However, this suggestion must be tempered by the possibility that these requirements may be properties of isolated enzymes and may not fully reflect the priorities of the enzymes within functional transcription complexes.

Thus, these experiments raise the interesting possibility that there may be three independent activities associated with polymerase II transcription that hydrolyze the beta- bond of ATP:promoter melting, CTD kinase, and DNA helicase. We have characterized that the first ATP dependent activity is involved in the critical promoter opening step. Future experiments will be required to learn the relationship of the other two activities to the pathway of mammalian mRNA transcription.


FOOTNOTES

*
This research was supported by National Institutes of Health Grant GM49048 and National Science Foundation Grant MCB92-03293 (to J. D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 310-825-1620; Fax: 310-825-0982.

(^1)
The abbreviations used are: pol II, polymerase II; AMP-PNP, 5`-adenylyl-beta,-imidodiphosphate; ATPS, adenosine 5`-O-(thiotriphosphate); CTD, C-terminal domain.

(^2)
Poljak and Gralla, unpublished observations.


REFERENCES

  1. Bunick, D., Zandomeni, R., Ackerman, S., and Weinmann, R. (1982) Cell 29, 877-886 [Medline] [Order article via Infotrieve]
  2. Carey, M., Leatherwood, J., and Ptashne, M. (1990a) Science 247, 710-712 [Medline] [Order article via Infotrieve]
  3. Carey, M., Lin, Y.-S., Green, M. R., and Ptashne, M. (1990b) Nature 345, 361-364 [CrossRef][Medline] [Order article via Infotrieve]
  4. Carpousis, A. J., and Gralla, J. D. (1985) J. Mol. Biol. 183, 165-177 [Medline] [Order article via Infotrieve]
  5. Conaway, R. C., and Conaway, J. W. (1988) J. Biol. Chem. 263, 2962-2668 [Abstract/Free Full Text]
  6. Conaway, R. C., and Conaway, J. W. (1993) Annu. Rev. Biochem. 62, 161-190 [CrossRef][Medline] [Order article via Infotrieve]
  7. Corden, J. L. (1990) Trends Biochem. Sci. 15, 383-387 [CrossRef][Medline] [Order article via Infotrieve]
  8. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475-1489 [Abstract]
  9. Feaver, W. J., Gileadi, O., Li, Y., and Kornberg, R. D. (1991) Cell 67, 1223-1230 [Medline] [Order article via Infotrieve]
  10. Gamper, H. B., and Hearst, J. E. (1982) Cell 29, 81-90 [Medline] [Order article via Infotrieve]
  11. Goodrich, J. A., and Tjian, R. (1994) Cell 77, 145-156 [Medline] [Order article via Infotrieve]
  12. Guzder, S. N., Sung, P., Bailly, V., Prakash, L., and Prakash, S. (1994) Nature 369, 578-581 [CrossRef][Medline] [Order article via Infotrieve]
  13. Jiang, Y., and Gralla, J. D. (1993) Mol. Cell. Biol. 13, 4572-4577 [Abstract]
  14. Jiang, Y., Smale, S., and Gralla, J. D. (1993) J. Biol. Chem. 268, 6535-6540 [Abstract/Free Full Text]
  15. Jiang, Y., Triezenberg, S., and Gralla, J. D. (1994) J. Biol. Chem. 269, 5505-5508 [Abstract/Free Full Text]
  16. Jiang Y., and Gralla, J. D. (1994) Nucleic Acids Res., in press
  17. Kim, Y. J., Bjorklund, S., Li, Y., Sayre, M. H., and Kornberg, R. D. (1994) Cell 77, 599-608 [Medline] [Order article via Infotrieve]
  18. Kirkegaard, K., Buc, H., Spassky, A., and Wang, J. C. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2544-2548 [Abstract]
  19. Koleske, A. J., and Young, R. A. (1994) Nature 368, 466-469 [CrossRef][Medline] [Order article via Infotrieve]
  20. Lu, H., Zawel, L., Fisher, L., Egly, J. M., and Reinberg, D. (1992) Nature 358, 641-645 [CrossRef][Medline] [Order article via Infotrieve]
  21. Meiklejohn, A. L., and Gralla, J. D. (1989) J. Mol. Biol. 207, 661-673 [Medline] [Order article via Infotrieve]
  22. Parvin, J. D., and Sharp, P. A. (1993) Cell 73, 533-540 [Medline] [Order article via Infotrieve]
  23. Pruss, G. J., and Drlica, K. (1989) Cell 56, 521-523 [Medline] [Order article via Infotrieve]
  24. Sasse-Dwight, S., and Gralla, J. D. (1989) J. Biol. Chem. 264, 8074-8081 [Abstract/Free Full Text]
  25. Sawadogo, M., and Roeder, R. G. (1984) J. Biol. Chem. 259, 5321-5326 [Abstract/Free Full Text]
  26. Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J. H. J., Chambon, P., and Egly, J.-M. (1993) Science 260, 58-63 [Medline] [Order article via Infotrieve]
  27. Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7476-7480 [Abstract]
  28. Serizawa, H., Conaway, R. C., and Conaway, J. W. (1993) J. Biol. Chem. 268, 17300-17308 [Abstract/Free Full Text]
  29. Siebenlist, U., Simpson, R. B., and Gilbert, W. (1980) Cell 20, 269-281 [Medline] [Order article via Infotrieve]
  30. Timmers, H. T. (1994) EMBO J. 13, 391-399 [Abstract]
  31. Wang, W., Carey, M., and Gralla, J. D. (1992a) Science 255, 450-453 [Medline] [Order article via Infotrieve]
  32. Wang, W., Gralla, J. D., and Carey, M. (1992b) Genes & Dev. 6, 1716-1727
  33. Young, R. A. (1991) Annu. Rev. Biochem. 60, 689-715 [CrossRef][Medline] [Order article via Infotrieve]
  34. Zawel, L., and Reinberg, D. (1993) Prog. Nucleic Acids Res. Mol. Biol. 44, 67-108 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.