A negative elongation factor for human RNA polymerase II inhibits the anti-arrest transcript-cleavage factor TFIIS Murali Palangat*, Dan B. Renner†, David H. Price†, and Robert Landick*‡ *Department of Bacteriology, University of Wisconsin, Madison, WI 53706; and †Department of Biochemistry, University of Iowa, Iowa City, IA 52242 Edited by Jeffrey W. Roberts, Cornell University, Ithaca, NY, and approved August 9, 2005 (received for review December 16, 2004)
Formation of productive transcription complexes after promoter escape by RNA polymerase II is a major event in eukaryotic gene regulation. Both negative and positive factors control this step. The principal negative elongation factor (NELF) contains four polypeptides and requires for activity the two-polypeptide 5,6-dichloro-1-D-ribobenzimidazole-sensitivity inducing factor (DSIF). DSIF兾 NELF inhibits early transcript elongation until it is counteracted by the positive elongation factor P-TEFb. We report a previously undescribed activity of DSIF兾NELF, namely inhibition of the transcript cleavage factor TFIIS. These two activities of DSIF兾NELF appear to be mechanistically distinct. Inhibition of nucleotide addition requires >18 nt of nascent RNA, whereas inhibition of TFIIS occurs at all transcript lengths. Because TFIIS promotes escape from promoter-proximal pauses by stimulating cleavage of backtracked nascent RNA, TFIIS inhibition may help DSIF兾NELF negatively regulate productive transcription. transcription elongation 兩 pausing 兩 backtracking
R
egulation of productive mRNA chain synthesis by RNA polymerase II (RNAPII) occurs when the RNA chain is ⬇10–100 nt long and determines whether RNAPII forms a fully functional transcription elongation complex (TEC) or halts RNA synthesis during the early stages of transcript elongation (reviewed in refs. 1–5). Conversion to a productive TEC involves an ordered set of transitions that require successive phosphorylation of Ser-5 and Ser-2 in the RPB1 C-terminal heptapeptide repeat domain (CTD) by kinase components of TFIIH and positive elongation factor P-TEFb, respectively. These CTD changes orchestrate release of initiation factors, recruitment of general elongation and chromatin-modifying factors, and capping of the nascent transcript by the capping enzyme. Completion of these steps is required to produce a TEC able to transcribe through nucleosomes on a chromatin template and synthesize full-length pre-mRNAs. During this process, negative elongation factor (NELF) is recruited to nascent TECs through interactions with the 5,6dichloro-1--D-ribobenzimidazole-sensitivity-inducing factor (DSIF) and RNAPII. DSIF is a heterodimer of SPT4 and SPT5 that alone can stimulate transcript elongation in some conditions (6, 7). NELF is a complex of four subunits: the Wolf–Hirschhorn syndrome candidate protein 2 (NELF-A), a cofactor of BRCA1 (NELF-B), one of two TH1-like products of alternatively spliced mRNAs (NELF-C兾D), and an RNA-binding protein, RD (NELF-E). A region of NELF-A that resembles hepatitis ⭸ antigen binds RNAPII (8), whereas a C-terminal region of RD (NELF-E) binds RNA (9) and is proposed to mediate inhibition of transcription through interaction with nascent RNA as the RNA emerges from the RNAPII exit channel (8). Both SPT5 and RD (NELF-E) also can be phosphorylated by P-TEFb (10–12), which may contribute to the formation of productive TECs. Although DSIF兾NELF interacts with RNAPII and RNA and slows or arrests extension of transcripts in vitro (13, 14), neither the fundamental mechanism by which it inhibits nucleotide 15036 –15041 兩 PNAS 兩 October 18, 2005 兩 vol. 102 兩 no. 42
addition nor the regulatory role of DSIF兾NELF inhibition of RNAPII is fully understood. NELF-induced slowing or arrest of nascent TECs may both provide time for productive TECgenerating steps like mRNA capping and serve as a checkpoint to block RNA synthesis in the absence of regulatory input (12, 15, 16). For instance, DSIF兾NELF induces promoter-proximal pausing in the Drosophila hsp70 gene (15), which is relieved upon heat shock through interactions of a heat-shock factor with the TEC (17). To allow productive transcription, NELF may be inactivated or released when P-TEFb phosphorylates SPT5, RD (NELF-E), and the RNAPII CTD (12, 14, 18); capping enzyme, which binds DSIF and the CTD, may help inactivate NELF (19). The transcript cleavage factor TFIIS is another participant in early elongation. TFIIS promotes productive elongation by stimulating cleavage of backtracked transcripts that arise as RNAPII enters and resides in pause and arrest states during early elongation, including promoter-proximal pauses in the Drosophila hsp70 gene (20, 21). Similarly, the bacterial factor GreA is required for the escape of RNAP from promoterproximal pauses upon recruitment of the positive elongation factor Q (22). Both TFIIS and GreA stimulate cleavage of backtracked RNAs by inserting in the RNAP secondary channel and stabilizing the binding of the second catalytic Mg2⫹ ion in the RNAP active site (Mg2⫹II; refs. 23–26). Thus, we wondered whether the inhibitory effect of DSIF兾NELF on productive elongation might in part involve counteracting the stimulatory effect of TFIIS on formation of productive TECs. To investigate this possibility, we examined the effects of DSIF兾NELF and TFIIS separately and together on TECs halted in the early transcribed region of HIV-1 in vitro. Materials and Methods Template DNA. DNA templates with upstream biotin tags were prepared by PCR amplification of plasmid pLL283 or pPM151A (truncated HIV-1 LTR) and immobilized on streptavidinagarose beads (Sigma), as described (27, 28). pPM151A was derived from pLL283 by deleting DNA coding for A15 through G48 of the HIV-1 transcript (template 2; Fig. 1A), yielding a truncated transcript with the pause site located at position ⫹U28. Transcription Factors. Human transcription factor TFIIS was over-
expressed in Escherichia coli and purified as described (29). DSIF and NELF were purified as described (14). Transcription Reactions and Pause Strength Calculations. U14 TECs
were prepared, converted to TECs with longer RNAs, and used This paper was submitted directly (Track II) to the PNAS office. Abbreviations: DSIF, 5,6-dichloro-1--D-ribobenzimidazole-sensitivity inducing factor; NELF, negative elongation factor; RNAPII, RNA polymerase II; TEC, transcription elongation complex; CTD, C-terminal heptapeptide repeat domain. ‡To
whom correspondence should be addressed. E-mail:
[email protected].
© 2005 by The National Academy of Sciences of the USA
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Fig. 1. Comparison of effects of DSIF兾NELF on TFIIS-stimulated transcript cleavage and on nucleotide addition. (A) Transcription templates. Sequences are shown for template 1 (wild-type HIV-1 transcript with the pause site indicated at U62) and template 2 (truncated HIV-1 transcript lacking anti-TAR and TAR with the pause site indicated at U28). Anti-TAR, dotted overline; TAR, black underline; truncation, open box. (B) Halted C27, U28, and U14 complexes (⬇10 pM) were incubated with DSIF (⬇60 nM) and NELF (⬇7 nM) for 2 min at 30°C before addition of TFIIS (⬇50 nM). Only a fraction of DSIF was active (14). Samples were removed at 8-s intervals and separated on a 12.5% denaturing gel (see Materials and Methods). The plots below each gel show the disappearance of C27, U28, and U14 RNA (F, ⫹DSIF兾NELF; E, ⫺DSIF兾NELF). (C) TECs halted at U11–U14 on template 1 were elongated with all four NTPs (1 mM each). Aliquots were removed at 7-s intervals; a final sample (lane C) was removed 4 min after raising GTP to 5 mM. RNA products were analyzed as described (28), and the fraction U14 RNA with error analysis from three replicate experiments was plotted against reaction time. The entire gel is shown in Fig. 5, which is published as supporting information on the PNAS web site. (D) TECs halted at A26 formed on template 2 were elongated through the pause site in the presence of CTP, GTP, and UTP. Samples were collected, processed, and analyzed as described for C. (E) Ratio of pause strengths (product of pause duration and pause efficiency; refs. 30 and 31) at different positions on template 1 in the presence and absence of DSIF兾NELF represented as a bar graph. Pause strengths correspond to the areas under the curves in C and D and were calculated (Materials and Methods) for full time courses and RNA electrophoretograms, an example of which is shown in Fig. 5.
Transcript Cleavage Reactions. TECs halted at U14, U16, C27,
U28, or U62 were incubated with TFIIS in the absence or presence of DSIF兾NELF at concentrations and for times indicated in the figure legends. Samples were processed, separated, and quantitated as described (28). Mn2⫹-induced intrinsic transcript cleavage was performed on U28 TECs that were washed with transcription buffer containing MnCl2 (10 mM) instead of Palangat et al.
MgCl2. Pyrophosphorolysis was performed by incubating U28 TECs with PPi (1 mM) for 5 min at 30°C. Results DSIF兾NELF Exhibits Two Independent Activities: Inhibition of Nucleotide Addition and of TFIIS. To test for an effect of DSIF兾NELF on
TFIIS action, we prepared halted TECs on two HIV-1-derived templates and then compared effects on TFIIS-mediated transcript cleavage with effects on transcript elongation at the same template positions. (TFIIS stimulates transcript cleavage in many halted or paused TECs but with release of smaller 3⬘ RNA fragments than from arrested TECs; ref. 3.) One template contained the promoter and early transcribed region of HIV-1, including a well characterized pause site at U62 known to form TECs susceptible to TFIIS-stimulated transcript cleavage (template 1, Fig. 1 A; refs. 27 and 28); the other (template 2) contained a deletion of sequences coding for the TAR (Transactivation responsive) and anti-TAR RNA structures. These PNAS 兩 October 18, 2005 兩 vol. 102 兩 no. 42 兩 15037
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for transcription assays as described. (27). Pause strengths (Fig. 1E) were calculated by sampling RNAs present at time intervals after addition of 1 mM NTPs to a set of initially formed TECs halted and washed at and before U14 (30). We prepared plots of the relative amounts of RNA vs. time (minus RNAs that had not reached the site in the final time point; plots similar to those in Fig. 1 C and D). Relative pause strength was calculated as the area under the line for each RNA species in the presence of DSIF兾NELF divided by the area under the line in the absence of DSIF兾NELF.
RNA structures influence, but are not required for, pausing (28). Thus, paused TECs still form at U28 of template 2. DSIF兾NELF inhibited TFIIS-stimulated transcript cleavage in different halted TECs (C27 and U28 on template 2; U14 on template 1; Fig. 1B). We assayed TFIIS using halted TECs to avoid complications of simultaneous nucleotide addition and transcript cleavage but verified that transcript-cleaved TECs remained elongation-competent with or without DSIF兾NELF when NTPs were added (data not shown). We observed similar DSIF兾NELF inhibition of TFIIS cleavage with G12, U16, and U62 TECs on template 1 or 2 (data not shown). Although the rate of TFIIS cleavage varied among TECs (from ⬇0.02 s⫺1 for U28 TEC to ⬇0.075 s⫺1 for C27 TEC at ⬇50 nM TFIIS), the magnitude of the DSIF兾NELF inhibitory effect was relatively constant for all TECs (3.4 ⫾ 0.15-fold at ⬇7 nM DSIF兾NELF). This result is consistent with DSIF兾NELF inhibiting by constant amounts in either TFIIS binding to RNAPII or TFIIS stimulation of cleavage, whereas the basal rate of TFIIS-stimulated cleavage is determined by the RNA sequence in the active site and the extent of TEC backtracking, which vary among TECs. In contrast to the constant effect of DSIF兾NELF on TFIISstimulated transcript cleavage, DSIF兾NELF exhibited variable effects on the rate of nucleotide addition. DSIF兾NELF actually accelerated nucleotide addition at U14, whereas it inhibited nucleotide addition at both C27 and U28 (Figs. 1 C and D and 5). This result is consistent with the idea that DSIF兾NELF inhibition of nucleotide addition involves interaction of RD (NELF-E) with nascent RNA (8, 9), because the U14 RNA should be entirely sequestered within RNAPII and unavailable for interaction with DSIF兾NELF. Previous reports suggest inhibition by DSIF兾NELF requires a minimum length of nascent RNA (13, 14, 19). We conclude that DSIF兾NELF possesses two different inhibitory modes. One mode, described previously, inhibits nucleotide addition when nascent RNA is approximately ⱖ18 nt. The second mode, described here, inhibits TFIISstimulated transcript cleavage irrespective of transcript length. To ask at which transcript length DSIF兾NELF begins inhibiting nucleotide addition, we examined the effect of DSIF兾NELF on transcript elongation at all positions after G11 on template 1. We calculated the apparent pause strength at different template positions and then plotted the ratio of apparent pause strengths with and without DSIF兾NELF as a function of template position (Fig. 1E; see Materials and Methods). Pause strength is the product of pause duration multiplied by pause efficiency but is simpler to measure than pause duration or efficiency individually (30, 31). When the nascent transcript was 17 nt or shorter, DSIF兾NELF reduced relative pause strength (i.e., increased the rate of transcription), whereas when the transcript was 18 nt or longer, DSIF兾NELF increased relative pause strength (i.e., decreased the rate of transcription). This result suggests that DSIF兾NELF commences inhibition when the RNA chain is 18 nt, which is roughly the position at which nascent RNA emerges from RNAPII (see Discussion). DSIF兾NELF acceleration of RNAPII when RNA is ⱕ17 nt may be caused by the previously reported stimulatory activity of DSIF (6). Both DSIF and NELF Are Required to Inhibit TFIIS. Enhancement of pausing by DSIF兾NELF requires both the DSIF and NELF components (14). To ask whether inhibition of TFIIS also depended on both NELF and DSIF, we tested them alone and in combination. Even at the same concentrations that inhibited TFIIS when combined, DSIF and NELF had no effect on TFIIS-stimulated cleavage when added individually to U28 TECs (Fig. 2A). Thus, like the stimulation of pausing, the inhibition of TFIIS by DSIF兾NELF requires both factors. Importantly, this result also demonstrates that TFIIS inhibition is a functional consequence of DSIF兾NELF interaction 15038 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0409405102
Fig. 2. Inhibition of TFIIS requires both DSIF and NELF and appears to be competitive. (A) Halted U28 complexes were incubated with either DSIF (⬇60 nM) or NELF (⬇7 nM) or both for 2 min at 30°C before addition of TFIIS (⬇50 nM) and removal of samples at 8-s intervals. Samples were separated on a 12.5% denaturing gel. (B) Competitive inhibition of TFIIS by DSIF兾NELF. TFIISmediated transcript cleavage of C27 TECs was performed as in A with the indicated amounts of TFIIS in the presence of DSIF (⬇9 nM) and NELF (⬇3.5 nM). The rate of cleavage of C27 TECs (in s⫺1) is plotted as a function of TFIIS concentration (F, ⫹DSIF兾NELF; E, ⫺DSIF兾NELF). (C) Increasing DSIF兾NELF concentration does not increase TFIIS inhibition. Rates of transcript cleavage of C27 TECs were determined as described in the legend to B. TFIIS was at 500 nM. 1⫻ DSIF was ⬇9 nM; 1⫻ NELF was ⬇3.5 nM.
and not an adventitious effect of a contaminant in DSIF or NELF preparations. DSIF兾NELF Inhibits TFIIS Binding. In principle, DSIF兾NELF could
inhibit TFIIS either by interfering with binding of TFIIS to the TEC or by altering the activity of the TEC so that it is less Palangat et al.
DSIF兾NELF Does Not Inhibit the Intrinsic Cleavage Activity of RNAPII.
If DSIF兾NELF did inhibit TFIIS-stimulated transcript cleavage by interfering with the catalytic mechanism rather than with TFIIS binding, DSIF兾NELF also should inhibit intrinsic transcript cleavage by RNAPII. Although TFIIS stimulates transcript cleavage by stabilizing Mg2⫹II binding, RNAPII can catalyze the same cleavage reaction without TFIIS at slower rates (33). The rate of intrinsic cleavage can be increased by high pH, substitution of Mn2⫹ for Mg2⫹, or high concentrations of Mg2⫹ (26, 33, 34). We reasoned that substitution of Mn2⫹ for Mg2⫹ had the least potential to perturb RNAPII or DSIF兾 NELF. Therefore, we tested the effect of DSIF兾NELF on the rate of Mn2⫹-mediated intrinsic cleavage. DSIF兾NELF had no effect on Mn2⫹-mediated intrinsic cleavage when compared side by side with its effect of TFIIS-induced cleavage of the same U28 TECs (Fig. 3; Fig. 6, which is published as supporting information on the PNAS web site). DSIF兾NELF also had no effect on the rate of pyrophosphorolysis in U28 TECs (Fig. 3). These results confirm that DSIF兾NELF inhibits TFIIS action by interfering with TFIIS binding and not by affecting the catalytic activity of RNAPII. Discussion We report here that DSIF兾NELF possesses a previously undescribed activity, inhibition of TFIIS-mediated transcript cleavage, that does not require contact to nascent RNA and thus occurs by a different mechanism than the known inhibitory effect of DSIF兾NELF on nucleotide addition. DSIF兾NELF inhibition of TFIIS may have important implications for the regulation of early stages of transcript elongation by RNAPII. DSIF兾NELF Inhibits TFIIS Binding Far from Its Proposed Contact with Exiting RNA, Consistent with DSIF兾NELF Tethering to RNAPII. Our
results establish that DSIF兾NELF inhibits TFIIS-stimulated transcript cleavage and strongly suggest that DSIF兾NELF inhibits TFIIS binding to RNAPII. Inhibition of binding is likely because (i) the magnitude of TFIIS inhibition is constant even when the basal cleavage rate varies (Fig. 1B); (ii) raising the TFIIS concentration gradually overcomes DSIF兾NELF inhibition (Fig. 2B); and (iii) intrinsic transcript cleavage, which occurs Palangat et al.
Fig. 3. Intrinsic transcript cleavage activity of RNAPII is not inhibited by DSIF兾NELF. U28 TECs were subjected to Mn2⫹-induced intrinsic transcript cleavage at 10 mM MnCl2, TFIIS-induced cleavage (50 nM as a control), or pyrophosphorolysis with 1 mM sodium pyrophosphate (PPi) in the absence or presence of DSIF兾NELF. MnCl2 was replaced with 8 mM MgCl2 in the TFIIS and PPi reactions. The reaction conditions were otherwise as described in the legend to Fig. 2 A. Some lane-to-lane variation reflects loading differences caused by inhomogeneous sampling of the bead-immobilized TECs. The relative amounts of U28, C27, and A26 RNAs and the rates of U28 cleavage (0.04 ⫾ 0.007 min⫺1) are indistinguishable with or without DSIF兾NELF (Fig. 6).
by the same catalytic mechanism as TFIIS-stimulated cleavage, is not inhibited by DSIF兾NELF (Fig. 3). Our results do not establish how the six-polypeptide DSIF兾 NELF complex interferes with TFIIS binding. To consider this question, we compared possible sites of TFIIS competition to the proposed sites of DSIF兾NELF inhibition of nucleotide addition and of DSIF binding to RNAPII. DSIF兾NELF inhibition of nucleotide addition is thought to be mediated by interactions of RD (NELF-E) with nascent RNA (8, 9, 12). Our finding that DSIF兾NELF inhibition of nucleotide addition requires ⱖ18 nt of nascent RNA (Fig. 1E) is consistent with this view, because both nuclease protection (35) and structural modeling (Fig. 4A and refs. 36 and 37) suggest that 16–18 nucleotides of nascent RNA are protected within RNAPII. In contrast to its RNA length-dependent effect on nucleotide addition, however, DSIF兾NELF inhibited TFIIS even in TECs in which the nascent RNA had not yet emerged from RNAPII (e.g., U14 TEC, Fig. 1B). This difference suggests that TFIIS inhibition does not require RD subunit interaction with upstream RNA. Instead, DSIF兾NELF appears to interfere with TFIIS binding to RNAPII, which requires that DSIF兾NELF obscure or distort the TFIIS interaction site. The site of TFIIS binding to the TEC is far from the point of nascent RNA exit (Fig. 4A). TFIIS binds on the downstream face of RNAPII, where it inserts a Zn2⫹-ribbon domain into the secondary channel of RNAPII to stabilize binding of the second active-site Mg2⫹ ion required for the transcript cleavage reaction (23, 32). In contrast, the nascent RNA exits on the upstream face, opposite to and ⬎100 Å away from the TFIIS-binding site (Fig. 4A and refs. 36 and 37). At least three explanations for the widely separated sites of DSIF兾NELF action are possible. An elongated DSIF兾NELF could span the distance between the exiting RNA and the TFIIS-binding site; DSIF兾NELF could bind near the RNA exit channel and allosterically weaken the TFIIS-binding site; or DSIF兾NELF could fluctuate between these two locations, tethered to a site between them. All three explanations are consistent with DSIF binding to RPB7 (Fig. 4A), a location proposed because an archaeal SPT4 homologue (Sulfolobus acidocaldarius RpoE) is found fused to RPB7 (4, 38) and because SPT5 may PNAS 兩 October 18, 2005 兩 vol. 102 兩 no. 42 兩 15039
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proficient at transcript cleavage. For instance, a subunit of DSIF兾NELF could contact the same site of RPB1 that has been found to contribute to TFIIS binding (32), or DSIF兾NELF could interact near the active site of RNAPII and prevent TFIIS stabilization of Mg2⫹II binding. To address this question, we examined C27 TECs formed on the template 2 (Fig. 2B). In the absence of DSIF兾NELF, TFIIS gave a half-maximal cleavage rate at ⬇20 nM, suggesting that TFIIS binds C27 TECs with a Kd of ⬇20 nM (Fig. 2B; C27 TECs were present in amounts equivalent to ⬇10 pM RNAPII). When DSIF兾NELF was present at ⬇3.5 nM, inhibition of TFIIS was overcome at high TFIIS concentration (saturation at ⬇1 M TFIIS; apparent Kd ⬇275 nM TFIIS; Fig. 2B), as predicted for an effect on TFIIS binding. These results are inconsistent with models in which DSIF兾NELF noncompetitively alters TEC activity (because high TFIIS concentration overcomes inhibition) and in which DSIF兾 NELF blocks TFIIS binding to RNAPII by binding directly to TFIIS (because the inhibitory effects of DSIF兾NELF persist even when TFIIS is present in significant excess). Interestingly, the competition between TFIIS and DSIF兾NELF is asymmetric: a 6-fold increase in DSIF and NELF concentrations, individually or together, does not increase inhibition of TFIIS (Fig. 2C). This suggests DSIF兾NELF either binds to more than one site on RNAPII, only one of which overlaps the TFIIS-binding site, or inhibits TFIIS binding by distorting the TFIIS-binding site (see Discussion).
Fig. 4. Mechanism of DSIF兾NELF inhibition of TFIIS. (A) Location of TFIIS binding to a TEC. A yeast RNAPII TEC model based on the crystal structure of Gnatt et al. (37) and the nucleic-acid-scaffold model of Korzheva et al. (36) is depicted with TFIIS located as reported by Kettenberger et al. (23). Portions of RNAPII (Left) are rendered semitransparent to reveal RNA, DNA, and TFIIS in the internal channels of the enzyme. (Left) The approximate length of exiting RNA; (Right) the distance between the exiting RNA and TFIIS. The location at which RPB1 CTD emerges from RNAPII (mobile magenta worm), the proposed SPT5-binding site (blue worm; ref. 39) on the RPB7 subunit (gray worm), the weakly bound Mg2⫹ ion required for transcript cleavage (yellow sphere; Mg2⫹II), and TFIIS (orange worm) are shown in both Left and Right. RPB4 is omitted for clarity. (B) Model depicting how DSIF兾NELF inhibition of TFIIS could regulate promoter-proximal pausing. Double-headed arrow depicts possible NELF fluctuation (one of several ways to explain DSIF兾NELF competition for TFIIS binding; see text). Red X depicts backtrack-paused RNAPII active site.
interact with RNAPII through RPB7 (39). However, the failure of 6-fold more DSIF兾NELF to increase TFIIS inhibition even though TFIIS can outcompete DSIF兾NELF inhibition (Fig. 2 B and C) favors either tethering of DSIF兾NELF or a second low-affinity mode of TFIIS binding. Either simple or allosteric competition between TFIIS and DSIF兾NELF predicts an increased effect of DSIF兾NELF at higher DSIF兾NELF concentrations. Even if DSIF兾NELF allosterically distorts the TFIISbinding site, a reciprocal distortion of the DSIF兾NELF site should occur upon TFIIS binding. In contrast, if tethered DSIF兾NELF obscures TFIIS binding via a secondary weak contact, then the local concentration of DSIF兾NELF at the TFIIS-competitive site would be constant, and further increases in DSIF兾NELF concentration would have no effect until its bulk concentration approached this local concentration. Thus, tethering of DSIF兾NELF to RNAPII, possibly by SPT5 binding to RPB7, can explain both why competition between TFIIS and DSIF兾NELF is nonreciprocal and how DSIF兾NELF can affect distant parts of a TEC (exiting RNA and TFIIS contact). Alternatively, weak binding of high-concentration TFIIS could reflect a second low-affinity mode of TFIIS binding to TECs that are saturated with nonfluctuating DSIF兾NELF at all DSIF兾 NELF concentrations we tested. Inhibition of TFIIS by DSIF兾NELF May Contribute to Promoter-Proximal Pausing and Negative Regulation of Early Elongation. During the
transition from the initial transcription complex to a productive 15040 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0409405102
TEC, RNAPII is susceptible to premature termination (40). This susceptibility ends when P-TEFb phosphorylates targets including SPT5, RD (NELF-E), and Ser-2 in the RPB1 CTD, which inactivates NELF or dissociates it from the TEC (13, 14, 41, 42). Luse and coworkers (20, 43) have established that these initial transcription complexes are especially susceptible to prolonged pausing or arrest. Promoter-proximal pausing and susceptibility to arrest persist until the nascent RNAs are longer than ⬇50 nt and occur by backtracking of RNAP along the RNA and DNA chains, in part because initial TECs lack nascent RNA structures stable enough to prevent backtracking. Backtracking causes pausing because the RNA 3⬘ nucleotide is displaced from the active site and leads to arrest when 3⬘ RNA becomes trapped in the RNAPII secondary channel (also called the pore and funnel; refs. 44 and 45). This extensive promoter-proximal pausing兾arrest can be relieved by TFIIS-stimulated cleavage of the backtracked RNA (20). We suggest that DSIF兾NELF inhibition of TFIIS may contribute to control promoter-proximal pausing兾arrest and formation of productive TECs (Fig. 4B). Because RNAPII is prone to backtracking in the promoter-proximal region and may depend on TFIIS to avoid becoming trapped in backtracked states, inhibition of TFIIS by DSIF兾NELF may increase pausing and arrest simply by preventing TFIIS from rescuing backtracked RNAPII at the promoter-proximal sites (Fig. 4B). Thus, DSIF兾 NELF may negatively regulate the transition to productive elongation in part by inhibiting TFIIS. Palangat et al.
A role of DSIF兾NELF inhibition of TFIIS makes sense in vivo. DSIF兾NELF directly regulates RNAPII pausing兾arrest in the early transcribed regions of Drosophila hsp70 gene and in certain estrogen-responsive human genes (15, 16). Adelman et al. (21) recently reported that both efficient escape of RNAPII from promoter-proximal pauses in hsp70 and heat-shock induction of hsp70 require TFIIS. Thus, DSIF兾NELF could contribute to promoter-proximal RNAPII stalling in hsp70 in part by inhibiting TFIIS. This effect may contribute to the regulation of many genes transcribed by RNAPII, for which control of premature termination is a common theme. However, the complex interplay of regulators that control these steps remains to be elucidated. NELF and the initiation factor TFIIF also inhibit each other (14). TFIIF, which assists promoter escape (46), also inhibits TFIIS-stimulated cleavage
We thank Rachel Mooney and Scotty Kyzer for comments on the manuscript. This work was supported by grants from the National Institutes of Health (GM38660 to R.L. and GM35500 to D.H.P.).
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BIOCHEMISTRY
(47) but not TFIIS suppression of pausing (48). The points at which NELF can first act on TFIIF or TFIIS and at which NELF is released from the TEC are uncertain. NELF appears to be inactivated by the capping enzyme (19). Thus, DSIF兾NELF may delay RNAPII elongation into genes via multiple inhibitory interactions that increase promoter-proximal pausing (inhibition of TFIIF and nucleotide addition) and prevent reactivation of stalled TECs (inhibition of TFIIS) until a regulatory factor (e.g., P-TEFb or Drosophila heat-shock factor), capping enzyme, or both, binds to the TEC, relieves DSIF兾NELF inhibition, and allows TFIIS-stimulated transcript cleavage of backtracked RNA in the promoter-proximal, stalled TECs.
Palangat et al.
PNAS 兩 October 18, 2005 兩 vol. 102 兩 no. 42 兩 15041
