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Previous Article | Next Article 
Molecular and Cellular Biology, February 2002, p. 762-773, Vol. 22, No. 3
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.22.3.762-773.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215
Received 7 June 2001/ Returned for modification 10 July 2001/ Accepted 29 October 2001
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ABSTRACT |
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INTRODUCTION |
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The transcription reaction is a multistep process that minimally involves the RNA polymerase and any associated factors first binding to the promoter DNA. After this point, transcription initiates in the presence of nucleoside triphosphates (NTPs). Initiated complexes of all RNA polymerases are capable of abortive initiation, the steady-state production of very short RNA products over time (11, 12, 21, 25). These initiated complexes then undergo a transition referred to as promoter escape in which they transform into elongation complexes. Ternary elongation complexes are quite stable and proceed through the remainder of the transcription reaction to complete RNA synthesis.
Numerous studies have characterized stable elongation complexes in transcription by bacteriophage T7, E. coli, and eukaryotic RNA polymerases (10, 14, 16, 17, 29, 33). While these studies reveal the nature of elongation complexes, little is known about the transitions that occur during early transcription to form stable elongation complexes. For example, initiated complexes undergo an intricate metamorphosis that simultaneously involves the release of accessory factors, extension of the melted region of the DNA, formation of an RNA-DNA duplex, and direction of the newly transcribed RNA out of the polymerase, all with the goal of forming a stable elongation complex that will complete synthesis of the transcript (17, 18, 35). Although we know that such events occur during early transcription, a unified view of the specific transitions and conformational changes that ternary transcription complexes undergo during this critical point in the transcription reaction has not yet emerged.
We are interested in understanding the transitions that occur during the early steps of mRNA transcription. Toward this goal, we previously studied the kinetics of several steps in the human RNA polymerase II transcription reaction (19, 20). We arrived at a model for eukaryotic transcription by experimentally isolating five distinct steps (see Fig. 1A): preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation. These studies revealed a crucial transition, termed escape commitment, which occurs during early transcription after initiation and prior to promoter escape (19). Escape commitment is complete after a 4-nucleotide (nt) RNA is synthesized, which occurs within 10 s after addition of NTPs to assembled preinitiation complexes. Notably, after this transition is complete, complexes are stable and committed to proceeding forward through promoter escape and the remainder of the transcription reaction. Escape-committed complexes [RPEC · (4nt RNA)] decay quite slowly compared to the rate at which they form (19). Other biochemical studies of early RNA polymerase II transcription have also characterized transitions that occur upon synthesis of a 4-nt RNA. Timmers and colleagues observed a distinct change in the melted region of the DNA when a 4-nt RNA was synthesized (13). In addition, Luse and colleagues found that stable ternary complexes formed when an RNA of 4 nt was transcribed (3, 22).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]UTP (5 µCi per reaction), 1 mM ApC, 1 mM CpU, and 1 mM CpA. A general outline of the transcription reaction follows, with details and exceptions given in the figures and figure legends. The general transcription factors and RNA polymerase II were preincubated in buffer A for 2 min at 30°C (10 µl per reaction), after which promoter DNA in buffer A at 30°C (10 µl per reaction) was added. Proteins and DNA were incubated together for 10 min, at which point nucleotides were added. Transcription proceeded for 20 min at 30°C. Reactions were stopped with 100 µl of a stop solution containing 3.1 M ammonium acetate, 10 µg of carrier yeast RNA, and 15 µg of proteinase K. The samples were ethanol precipitated and resolved by 6% denaturing polyacrylamide gel electrophoresis (PAGE). For the experiments for which results are shown in Fig. 1C, 8B, and 8C, there were the following exceptions. Nucleotide concentrations were 1 mM ApC, 100 µM CTP, and 0.5 µM [-32P]UTP (5 µCi per reaction). Before the reactions were stopped, reaction mixtures were passed through G25 spin columns (Amersham Pharmacia) preequilibrated in buffer A. Eluates were heated for 3 min at 70°C and then treated with calf intestinal alkaline phosphatase (10 U) at 37°C for 20 min. Three microliters of stop mix containing 200 mM EDTA, 20% glycerol, and 0.025% bromophenol blue was added. Products were resolved by 20% denaturing PAGE and sized by UV shadowing 2-, 3-, 4-, and 5-nt RNA standards of the appropriate sequences. For the promoter-independent transcription assays for which results are shown in Fig. 6B and C, poly(dA-dT) · poly(dA-dT) (0.5 mg/ml; Sigma) was added to reaction mixtures in place of promoter DNA. The general transcription factors were omitted, and final nucleotide concentrations were 625 µM ATP and 25 µM [-32P]UTP (5 µCi per reaction).
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-32P]UTP (5 µCi per reaction). Reactions were stopped at varying time points (up to 10 min) with heating at 70°C for 3 min, and then reaction products were treated with calf intestinal alkaline phosphatase (10 U) at 37°C for 20 min. Three microliters of a stop solution containing 200 mM EDTA, 20% glycerol, and 0.025% bromophenol blue was added. A 6-µl volume of each reaction product was loaded on a 20% denaturing gel. The amount of product produced at each time point was quantitated using a Molecular Dynamics PhosphorImager and normalized with respect to time. For each condition tested, a parallel reaction was performed with a mutant AdMLP in which the start site position was changed from an A to a G (nontemplate strand) (11). The product produced from the mutant template originated from nonspecific start sites on the plasmid DNA and was subtracted as background from the product produced from the wild-type template. The sizes of the RNA products were confirmed by UV shadowing 3-nt RNA standards of the appropriate sequences. Electrophoretic mobility shift assays. Reactions were performed in the buffers described for in vitro transcription assays. RNA polymerase II was added to 32P-labeled oligonucleotides, as indicated in the figure legends. Binding occurred at 30°C for 10 min. Complexes were resolved on native 4% polyacrylamide gels (37.5:1 arylamide-bis-acrylamide, 5% glycerol, and 0.5x Tris-borate-EDTA) at 150 V for 3 h.
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RESULTS |
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Stable ternary complexes form only after synthesis of a 4-nt RNA. Although the experiment for which results are shown in Fig. 1B was performed under conditions in which the full-length RNA transcript was observed, it should be noted that stability to ctDNA occurs once a 4-nt RNA is synthesized (19). If escape commitment is the distinct point in transcription at which stable ternary complexes form, transcribing complexes prior to this point should not be stable. To directly address ternary complex stability both before and after synthesis of a 4-nt RNA, we performed the experiment for which results are shown in Fig. 1C. As diagrammed in the schematic, we utilized a mutant of the AdMLP (+5mt) in which the T at position +5 was changed to an A. By use of this mutant, a 4-nt RNA was the longest product that could be made when transcription was initiated with the dinucleotide ApC, as well as UTP and CTP. To assess ternary complex stability, reaction mixtures were passed through gel filtration spin columns so that only those RNAs stably bound in ternary complexes would be present in the eluate (22). As shown in Fig. 1C, an abundance of 3-nt RNA was detected in the reaction product that had not been passed through a spin column (lane 1). As shown in lane 2, however, 3-nt RNA was no longer detected after ternary complexes were passed through a spin column. This indicates that the 3-nt RNA we observed was produced abortively and released; thus, it is not part of stable complexes. Strikingly, a longer exposure of lane 2 revealed 4-nt RNA that survived the spin column as part of ternary complexes (Fig. 1C, lane 3). This confirms that stable ternary complexes form only after synthesis of a 4-nt RNA, concomitant with the completion of escape commitment.
The point at which escape commitment occurs depends on the length of the transcribed RNA. The point at which escape commitment is complete could be dictated by either the length of the transcribed RNA or the position of the polymerase active site with respect to the promoter DNA elements. To distinguish between these possibilities, we used the method shown in Fig. 2A, in which nucleotides were added to transcription reaction mixtures in two steps: limited nucleotides were added first, followed by remaining nucleotides 30 s later. Together, these nucleotides allowed synthesis of a full-length transcript; however, by altering the composition of the limited nucleotides, we controlled both the length of the RNA produced and the position of the polymerase on the template DNA during early transcription. We determined whether the limited nucleotides were sufficient for escape commitment to occur. As shown in Fig. 2B, transcription was initiated at either the -1, +1, or +2 position of the AdMLP and the polymerase was paused at different positions on the template DNA depending on which limited nucleotides were added. ctDNA was added either with the limited nucleotides (point 2) or with the remaining nucleotides (point 3). If the limited nucleotides were sufficient for escape commitment to occur, there was a ca. 10-fold increase in the level of transcript produced when ctDNA was added at point 3 compared to point 2, as reflected in the bar plot on the right. We found that irrespective of where transcription initiated and where the polymerase paused on the promoter DNA, escape commitment always occurred when an RNA of 4 nt or longer was made (reactions 2, 4, 5, 7, and 8) and did not occur when a 3-nt RNA was made (reactions 1, 3, and 6). These experiments showed that the length of the RNA transcript determines the point at which escape commitment is complete. Therefore, escape commitment depends on the distance between the active site of the polymerase and the 5' end of the RNA, not on the distance between the active site and promoter DNA elements such as the TATA box. This indicates that the growing transcript itself participates in a transition that occurs during early transcription.
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Single-stranded ends of ctDNA directly attack transcription complexes to block escape commitment. We reasoned that determining the property (or properties) of ctDNA responsible for inhibiting escape commitment would provide insight into the mechanism by which this step occurs. There are at least two models to explain the observation that ctDNA inhibits escape commitment. First, one of the general transcription factors might be released from the promoter during early transcription and would have to rebind for productive transcription to continue. ctDNA could sequester the released general transcription factor and prevent it from rebinding. Second, ctDNA might directly attack ternary complexes that contain all of the general transcription factors and inhibit a transition that occurs during escape commitment. For example, if one of the DNA or RNA binding grooves on RNA polymerase II is exposed, ctDNA could enter this site on the polymerase and block the transition to escape-committed complexes. To begin to distinguish between these possibilities, we tested whether a different nucleic acid inhibitor, poly(dI-dC) · poly(dI-dC), could inhibit escape commitment. Experiments were performed as diagrammed in Fig. 3A. As shown in Fig. 3B, poly(dI-dC), as opposed to ctDNA, was not able to inhibit escape commitment (compare lanes 2 and 3 with lanes 5 and 6). Poly(dI-dC) did, however, inhibit preinitiation complex formation completely when added with the AdMLP prior to addition of the general transcription machinery (Fig. 3B, lane 1). Furthermore, it inhibited each individual transcription factor from binding to the promoter after the others were prebound (data not shown). The observation that poly(dI-dC) cannot inhibit escape commitment but can prevent each protein from binding the promoter argues against protein release and rebinding as the mechanism by which ctDNA inhibits escape commitment. Instead we favor a model in which RNA polymerase II is directly attacked by ctDNA, and a change in ternary complexes during escape commitment renders the polymerase resistant to this attack.
We hypothesized that single-stranded ends generated during the preparation of sonicated ctDNA might be required for inhibition of escape commitment. To test this, we treated ctDNA with Klenow fragment (in the presence of dNTPs) to eliminate single-stranded 5' and 3' overhangs. The Klenow fragment-treated ctDNA did not inhibit escape commitment to the same extent as untreated ctDNA (Fig. 3C); hence, single-stranded ends are involved in inhibiting this step.
Identification of oligonucleotides that are potent inhibitors of escape commitment. Because the single-stranded ends of ctDNA were required to inhibit escape commitment, we sought to identify one or more single-stranded oligonucleotides that could inhibit escape commitment and mimic the effects of ctDNA. Doing so would provide an essential experimental tool to further probe the mechanism of escape commitment. We tested several oligonucleotides with both random and homopolymeric sequences by adding them to transcription reaction mixtures at points 1, 2, and 3 (see Fig. 3A). Figure 4A shows the effects of four different homopolymeric oligonucleotides 29 residues in length composed of either deoxyadenosines, deoxythymidines, deoxycytidines, or deoxyguanosines. We found that the 29-dC and 29-dG oligonucleotides inhibited escape commitment (Fig. 4A, lanes 8 and 9 and lanes 11 and 12, respectively), while the 29-dA and 29-dT oligonucleotides did not (lanes 2 and 3 and lanes 5 and 6, respectively). Moreover, the 29-dG oligonucleotide is a rather potent inhibitor of escape commitment: 95% inhibition was obtained with only 5 to 10 pmol of this oligonucleotide added to reaction mixtures at point 2 (Fig. 4B). The 50% inhibitory concentration (IC50) for the 29-dG oligonucleotide is 45 nM, while that for the 29-dC oligonucleotide is 6.7 µM.
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The oligonucleotide inhibitors target core RNA polymerase II. We hypothesized that single-stranded nucleic acids inhibit escape commitment by targeting RNA polymerase II and blocking the transition to escape-committed complexes. If so, the oligonucleotides that inhibit escape commitment should bind directly to RNA polymerase II. To test this, we performed an electrophoretic mobility shift assay (Fig. 5A). The 32P-labeled 20rG oligonucleotide formed a complex with RNA polymerase II (Fig. 5A, lane 2). When additional unlabeled oligonucleotide inhibitors were titrated into reaction mixtures, the complex was efficiently competed away only by the 20rG and 29-dG oligonucleotides. Hence, the oligonucleotides bind to RNA polymerase II with the same sequence specificity with which they inhibit escape commitment. Therefore, the sequence specificity with which the oligonucleotides inhibit transcription is likely dictated by their ability to bind to the polymerase.
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Poly(G) oligonucleotide inhibitors must be of sufficient length to inhibit escape commitment. We next asked whether the ability of poly(G) RNA to inhibit escape commitment depends on its length. We titrated an RNA oligonucleotide consisting of 10 guanosine residues (10rG) into transcription reactions prior to escape commitment (point 2 in Fig. 3A). As shown in Fig. 6A, the 10rG oligonucleotide was able to inhibit escape commitment only when added at levels much higher than those observed for the 20rG oligonucleotide (see Fig. 4D). The IC50 for the 10rG oligonucleotide is 0.5 µM, which is at least 40-fold greater than that observed for the 20rG oligonucleotide. Moreover, complete inhibition was never observed with the 10rG oligonucleotide. This finding indicates that inhibitor oligonucleotides must be of a certain length to effectively block escape commitment.
We questioned if the difference in the abilities of the 10rG and 20rG oligonucleotides to inhibit escape commitment reflected their relative affinities for binding RNA polymerase II. To directly test this, we performed electrophoretic mobility shift assays with RNA polymerase II and either the 32P-labeled 10rG oligonucleotide or the 32P-labeled 20rG oligonucleotide. RNA polymerase II bound each oligonucleotide with an approximate Kd of 
1.5 nM (Fig. 6B). Therefore, the >40-fold decrease in the ability of the 10rG oligonucleotide to inhibit escape commitment is not due to an impaired ability to bind to RNA polymerase II. We also asked whether the 10rG and 20rG oligonucleotides bind RNA polymerase II with similar kinetic stabilities. To test this, we monitored the rates at which the 10rG and 20rG oligonucleotides dissociated once bound to the polymerase. The respective oligonucleotides were prebound to RNA polymerase II; then a 500-fold excess of unlabeled oligonucleotide was added for various times. As shown in Fig. 6C, with both the 10rG and 20rG oligonucleotides, there was little reduction in the amount of 32P-labeled oligonucleotide bound to RNA polymerase II over the course of 1 h. As controls, lanes 2 and 8 show the amount of bound polymerase in the absence of unlabeled oligonucleotide, and lanes 3 and 9 show that the unlabeled oligonucleotides fully compete binding when added prior to RNA polymerase II. These data indicate that both oligonucleotides bind quite stably to RNA polymerase II.
The observation that the 10rG oligonucleotide cannot inhibit escape commitment but can bind tightly and stably to RNA polymerase II indicates that binding and inhibition are separable. This implies that the high-affinity site on the polymerase to which these oligonucleotides bind is removed from the site at which inhibition occurs. Because the inhibition of escape commitment is lost as the length of the oligonucleotide inhibitor is shortened, we favor a model in which the inhibitory oligonucleotides bind a distinct site on RNA polymerase II and then extend through the RNA exit groove toward the active site of the polymerase. The 20rG oligonucleotide reaches far enough through the exit groove to effectively inhibit escape commitment, whereas the 10rG oligonucleotide does not.
The 29-dG oligonucleotide does not block abortive synthesis of 3-nt RNA transcripts. To better understand the mechanism of escape commitment, we needed to determine the exact point in the transcription reaction at which the inhibitors act, and therefore at which escape commitment occurs. We have shown that all ternary complexes after synthesis of a 4-nt RNA were both stable and resistant to ctDNA (Fig. 1C and 2) (19). We demonstrated that initiated complexes producing 3-nt RNAs were unstable (Fig. 1C), but we were not certain whether the inhibitors could target these complexes. We therefore asked whether the 29-dG oligonucleotide inhibited abortive initiation, using a steady-state assay that monitors the synthesis of 3-nt transcripts produced specifically from the start site of the AdMLP (diagrammed in Fig. 7A). As shown in Fig. 7B, there was little difference in the amounts of trinucleotide product made over time in the presence or absence of the inhibitor. We also observed no inhibition by the 20rG oligonucleotide during the production of 3-nt RNA by core RNA polymerase II on poly(dA-dT) templates (data not shown). From these data several important conclusions can be drawn. First, the inhibitors do not block phosphodiester bond synthesis per se. Second, the inhibitors do not block nucleotide binding by the polymerase. Third, the inhibitors act after synthesis of a 3-nt RNA; thus, escape commitment does not begin until after this point.
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If escape commitment involves translocation after synthesis of the third phosphodiester bond, then a 4-nt RNA and nothing longer would be produced in the presence of the inhibitor. To test this, we determined the sizes of RNA products stably bound in transcribing complexes in the absence and presence of inhibitor. As shown in Fig. 8A, nucleotides sufficient to allow synthesis of a 15-nt RNA were added to preinitiation complexes with or without the 20rG oligonucleotide. Reaction mixtures were then passed through gel filtration spin columns to separate RNA bound in stable complexes from RNA produced abortively and released (22). As shown in Fig. 8B, in the absence of the inhibitor a 15-nt RNA was present in ternary complexes (lane 1). In the presence of the inhibitor, however, only a 4-nt product and nothing longer was observed (lane 2). This clearly demonstrates that the third phosphodiester bond can be synthesized in the presence of the inhibitor but subsequent phosphodiester bonds cannot. In these reactions transcription was initiated with the dinucleotide ApC, which lacks 5' phosphates. To determine if the presence of a 5' triphosphate would alter the point at which the inhibitor acted, we performed a similar reaction in which the initiating nucleotide was ATP rather than ApC. Again, only a 4-nt RNA and nothing longer was observed (lane 3). Under these conditions, in the absence of an inhibitor a 390-nt RNA was produced (data not shown). We conclude that the inhibitors block translocation after synthesis of a 4-nt RNA irrespective of the number of phosphates on the 5' end of the RNA transcript.
Lastly, we wanted to determine whether inhibited ternary complexes containing 4-nt RNAs were still catalytically active. This is important because it would demonstrate that the 20rG oligonucleotide does not cause a conformational change resulting in nonfunctional ternary complexes. Isolated escape-committed complexes cannot proceed forward to lengthen the 4-nt RNA because the 20rG oligonucleotide is still bound, given the slow off-rate (Fig. 6). Therefore, to assess catalytic activity, we asked whether the RNA polymerase II in the inhibited ternary complexes was capable of carrying out pyrophosphorolysis. It had been shown previously that stable RNA polymerase II ternary complexes can undergo pyrophosphorolysis in the presence of pyrophosphate (34). We used spin columns to isolate oligonucleotide-inhibited ternary complexes containing 4-nt RNAs. These were then incubated in the presence or absence of pyrophosphate. As shown in Fig. 8C, in the presence of pyrophosphate the 4-nt RNA disappeared, indicating that these complexes can carry out pyrophosphorolysis. Therefore, we can conclude that in blocking escape commitment the 20rG oligonucleotide does not inhibit catalysis per se.
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DISCUSSION |
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Model for the molecular mechanism of escape commitment by RNA polymerase II. The exact point at which the inhibitor oligonucleotides act, as well as where escape commitment occurs with respect to the remainder of the transcription reaction, is shown in Fig. 9. When preinitiation complexes are provided with nucleotides, transcription initiates. Initiation encompasses production of 2- and 3-nt RNA products, which can be abortively synthesized and released in the presence of the oligonucleotide inhibitors. After synthesis of a 4-nt RNA, but prior to translocation of the polymerase active site, the ternary complexes are susceptible to attack by the oligonucleotide inhibitors. Once the polymerase active site translocates to the position required for fourth phosphodiester bond synthesis, ternary complexes are escape committed and resistant to the addition of inhibitors. At this point, stable ternary complexes proceed forward through promoter escape, which occurs during synthesis of the 4th through the 14th phosphodiester bonds. We have previously reported that in the presence of ctDNA, the RNA polymerase II transcription reaction branches prior to completion of escape commitment (19). Here we developed new inhibitors that allowed us to determine that the transcription reaction branches when the polymerase active site attempts to translocate after synthesis of the third phosphodiester bond. We have also characterized the kinetic mechanism of the transcription reaction at the human interleukin-2 promoter. In these studies we found that escape commitment also occurs on the interleukin-2 promoter upon synthesis of a 4-nt RNA (9a). This suggests that escape commitment could be a generalizable step in early transcription by RNA polymerase II.
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The results we present here are significant because they demonstrate that translocation of the polymerase active site, and not phosphodiester bond synthesis, results in escape-committed complexes. Escape commitment also occurs independently of where the polymerase active site is located with respect to the promoter DNA. Our data show that RNA polymerase II senses the actual length of the transcript RNA during escape commitment as opposed to the number of phosphodiester bonds it has synthesized. Finally, the point of escape commitment is unaffected by the presence of phosphates on the 5' ends of the transcript. The proposal that escape commitment is accompanied by a conformational change agrees with previous biochemical studies of RNA polymerase II transcription. We and others have observed an increase in ternary complex stability after the synthesis of a 4-nt RNA (3, 22), and Timmers and colleagues observed a change in the melted region of the DNA at the same juncture in the reaction (13).
Recently, Kornberg and colleagues solved a crystal structure of a yeast RNA polymerase II elongation complex (10). The structure contains the multisubunit RNA polymerase II bound to a tailed DNA template from which the polymerase transcribed a 9-nt RNA. Within the ternary elongation complex, the position of the RNA transcript with respect to the active site of the polymerase reveals that the RNA polymerase has completed phosphodiester bond synthesis, but the active site has not yet translocated with respect to the RNA-DNA hybrid. Additionally, Kornberg and colleagues solved the crystal structure of free RNA polymerase II in the absence of any nucleic acids (7, 8). Comparing the structure of the elongation complex to that of the free polymerase suggests that conformational changes occur during early transcription. For example, a series of switch regions in the polymerase undergo conformational changes and folding transitions that are likely induced by binding to the downstream DNA and/or the DNA-RNA hybrid (10). The 4 nt on the 3' end of the RNA are buried in the transcribing complex and are in direct contact with the RNA polymerase (10). In considering the structure of the elongation complex, it was predicted that once translocation after production of a 4-nt RNA occurred, the 5' end of the RNA would become exposed for the first time. At this point one additional long-range interaction between R497 of Rpb2 and the 5' end of the RNA would take place (10). This is the exact point in the transcription reaction at which escape commitment occurs and stable ternary complexes form, and at which we predict a conformational change happens. The crystal structures suggest that changes in the polymerase might occur at this juncture; however, a detailed understanding of early transcription and how the conformations of ternary complexes change will require further biochemical studies and additional structural studies of ternary complexes trapped during early transcript synthesis.
A crystal structure by Cheetham and Steitz of a transcribing bacteriophage T7 RNA polymerase provided the first structural view of an initiated RNA polymerase and interestingly indicated that a transition at +4 occurs (5, 6). The conformations of the single-stranded promoter DNA, 3 nt of transcribed RNA in a heteroduplex with the DNA, and an incoming ribonucleoside triphosphate analog are clearly shown within the active-site pocket of the T7 RNA polymerase structure. The authors predict that extending the RNA-DNA duplex by even one additional base pair (i.e., to a total of 5 bp) would result in steric clashes between the 5' end of the RNA and the N-terminal domain of the polymerase. Thus, it appears that a critical conformational change during early transcription must also occur at register +4 in bacteriophage T7 RNA polymerase for transcription to continue. Several studies of early transcription by E. coli RNA polymerase have also observed distinct transitions after synthesis of a 4-nt RNA (2, 9, 31). This juncture is the precise point in the eukaryotic RNA polymerase II transcription reaction where we found escape commitment to occur. Determining whether these transitions are related to escape commitment by RNA polymerase II will require future studies.
Mechanism by which inhibitor oligonucleotides block escape commitment. We identified two oligonucleotides, the 20rG oligonucleotide and the 29-dG oligonucleotide, that potently inhibit escape commitment by binding directly to RNA polymerase II. It is not yet clear why a strong preference for guanosine residues exists. We predict that the 20rG and 29-dG oligonucleotides bind to a distinct site on the polymerase and extend through the RNA groove that exits the active-site pocket of the polymerase. This is based on (i) the dependence on RNA transcript length for completing escape commitment (Fig. 2), (ii) the potency with which the RNA oligonucleotide inhibits (Fig. 4), and (iii) the observation that inhibitor oligonucleotides must be of sufficient length to block escape commitment (Fig. 6). Our findings show that 10 guanosine residues are sufficient for high-affinity binding to the polymerase but that to block escape commitment the oligonucleotide must be of sufficient length to extend into the region of the ternary complex at which inhibition occurs (perhaps by channeling through the RNA exit groove). The inhibitor could then block a conformational change during escape commitment, thereby preventing productive transcription from continuing. We know that the conformation of the inhibited complex is quite stable, because it retains the 4-nt RNA for long periods of time and survives passage through a spin column. We also know that the inhibited complex is still active, because it is able to carry out pyrophosphorolysis. We are not yet certain, however, whether these inhibited complexes could recover and begin productive transcription again if the inhibitor were removed. Johnson and Chamberlin previously observed that RNA oligonucleotides could bind to yeast RNA polymerase II in a manner that mimicked a ternary complex (15). These interactions did not inhibit transcription, did not exhibit sequence specificity, and did not exhibit low nanomolar Kds; thus, they are likely distinct from the interactions reported here. Chamberlin and colleagues also found that E. coli RNA polymerase has two RNA binding sites that are thought to bind the elongating transcript (1, 27). If analogous sites exist on RNA polymerase II, one or both could be responsible for binding the oligonucleotide inhibitors.
The oligonucleotide inhibitors we described here inhibit a specific translocation event during early transcription. At a glance, this appears similar to the mechanism by which the antibiotic rifampin (RIF) inhibits transcription by bacterial RNA polymerases; however, the inhibition we observe is quite different from that observed for RIF. Most significantly, the step blocked by the inhibitors characterized here (i.e., escape commitment) is a critical transition that occurs naturally during early transcription by RNA polymerase II. Escape commitment results in stable ternary complexes and is a step that can be controlled by the helicase activity of TFIIH (3, 13, 19, 22). In contrast, the point at which RIF blocks transcription from continuing, as described in more detail below, is not considered to be a unique transition during early transcript synthesis by bacterial RNA polymerases. RIF blocks translocation during early transcription at a point dependent on the nature of the phosphate group(s) on the 5' end of the transcript RNA. Transcripts initiated with NTPs are blocked after formation of a 2-nt RNA, whereas transcripts initiated with dinucleotides or nucleoside di- or monophosphates are blocked after synthesis of a 3-nt RNA (4, 26). Our data, however, demonstrate that inhibition of escape commitment by oligonucleotides does not depend on the number of phosphates on the 5' end of the RNA transcript (Fig. 8). Moreover, when RIF blocks translocation during early transcription by bacterial RNA polymerases, the short RNA products that form are produced and released. In contrast, when the 20rG oligonucleotide inhibits escape commitment, the 4-nt RNA remains stably bound in ternary complexes. Finally, inhibition of escape commitment depends on the length of the single-stranded oligonucleotide inhibitor (Fig. 6). This supports a model in which the inhibitor oligonucleotides bind to RNA polymerase II at a site removed from the region at which inhibition occurs, as opposed to binding a RIF-like pocket near the region of inhibition. Although RIF and the oligonucleotide inhibitors both block translocation events, the mechanisms by which they do so differ.
Biochemical and structural data from several different RNA polymerases indicate that critical conformational changes likely occur during early transcription in order to facilitate the transition from an unstable initiation complex to a stable elongation complex. Our data suggest that one such change occurs during escape commitment as the RNA polymerase II active site translocates after synthesis of the third phosphodiester bond.
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ACKNOWLEDGMENTS |
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This research was supported by Research Project Grant RPG-00-271-01-MGO from the American Cancer Society. J.A.G. is a Pew Scholar in the Biomedical Sciences.
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FOOTNOTES |
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