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RNA polymerase II elongation factor: Biological Overview | References

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. This study reports that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, it was discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction (Adelman, 2005; full text of article).

In a search for elongation factors that directly affect the heat shock response, a role for the transcript cleavage factor TFIIS was investigated. Like the bacterial Gre factors, TFIIS rescues RNA polymerase that has undergone reverse translocation, or 'backtracking' along the DNA template (Borukhov, 1993; Fish, 2002, Wind, 2000). Backward movement misaligns the 3' end of the nascent RNA with the RNA polymerase active site, thereby prohibiting continued RNA synthesis (Komissarova, 1997). Transcript cleavage factors restart the arrested RNA polymerase by inducing internal cleavage of the RNA by the polymerase active site, creating a new 3' end that is properly aligned for catalysis. The activity of transcript cleavage factors has been reported to stimulate promoter escape and transcription elongation and to decrease pausing. Recently published structural and functional analyses of transcript cleavage factors GreB and TFIIS complexed with their respective RNA polymerases elucidate the mechanism of this activity (Kettenberger, 2003; Laptenko, 2003; Opalka, 2003 and Sosunova, 2003): TFIIS inserts a long coiled-coil domain into the RNA polymerase secondary channel, helping to coordinate a Mg+2 ion required for the reverse-catalytic reaction. However, although the detailed mechanism of TFIIS activity is known, the in vivo roles for this activity remain poorly defined (Adelman, 2005).

To test whether Pol II complexes stalled within the promoter-proximal region were inactive due to transcription arrest, whether they could be rescued by transcript cleavage factor TFIIS was investigated. Stalled early elongation complexes (EEC) formed in a partially fractionated embryo extract lacking TFIIS were isolated and washed before restarting transcription in the presence or absence of purified TFIIS. The data show that the addition of NTPs leads to little or no transcription elongation in the absence of TFIIS. The presence of purified TFIIS alone induced efficient cleavage of RNA products associated with stalled Pol II. The sensitivity of specific RNAs to TFIIS-dependent cleavage signifies that these RNA species are associated with backtracked, arrested Pol II complexes. Cleavage of these RNAs in the presence of TFIIS reactivates the stalled complexes, allowing the labeled RNA species to be elongated upon addition of NTPs. It is concluded that TFIIS-induced cleavage rescues the promoter-proximal stalled, arrested Pol II (Adelman, 2005).

Taken together, these data suggest that intrinsic pause sites within the promoter-proximal region of hsp70 are recognized in vitro, perhaps with the aid of regulatory elongation factors, and that Pol II at these locations rapidly become inactive. However, the experiments demonstrating transcription arrest involve EEC that were artificially stalled and stringently washed prior to analysis, which does not accurately reflect the dynamics of hsp70 transcription. Thus, to investigate whether Pol II actively transcribing through the promoter-proximal region is susceptible to arrest and to determine the role of TFIIS in this process, a transcription assay was performed in a fractionated Drosophila embryo extract that lacked TFIIS (Adelman, 2005).

EEC were radiolabeled during elongation to position +16 nt and washed thoroughly with transcription buffer plus heparin to remove unincorporated NTPs and unbound extract proteins and to prevent reinitiation. The resulting EEC were split into two equivalent reactions, one of which was supplemented with purified TFIIS. Unlabeled NTPs were added to restart transcription, and aliquots were removed various time points. In the absence of TFIIS, Pol II accumulated in the promoter-proximal region and was not able to escape from sites of stalling during the time course. In contrast, inactive Pol II complexes were barely detectable in the presence of TFIIS. Instead, TFIIS stimulated rapid and efficient elongation of the labeled +16 nt RNA through the promoter-proximal region, leading to the formation of increased levels of full-length transcript. TFIIS activity also generated cleavage products that were released from Pol II. These data indicate that the initially transcribed sequence of hsp70 contains intrinsic sites at which Pol II pauses or stalls during active transcription, and that TFIIS is critical for efficient elongation through this region (Adelman, 2005).

To verify the functional relevance of TFIIS in the heat shock response in vivo, TFIIS levels were depleted in Drosophila S2 cells using RNAi. S2 cells that were untreated or treated with dsRNA targeting TFIIS were heat shocked to induce production of hsp70 RNA before harvesting cells and isolating total RNA. The depletion of TFIIS was not complete, perhaps due to the abundance or low turnover of the TFIIS protein; nonetheless, TFIIS-depleted cells were estimated to contain only ~10% of normal levels of TFIIS (Adelman, 2005).

Analysis of hsp70 RNA levels by quantitative RT-PCR reveals that TFIIS-depleted cells are indeed deficient in the heat shock response. In particular, there is a dramatic delay in hsp70 production in TFIIS-depleted cells, with hsp70 levels barely increasing above background after 2.5 min of heat shock. The significant kinetic block in hsp70 RNA production in TFIIS-depleted cells observed after a short heat shock, begins to be overcome at later time points, leading to an overall heat shock response of approximately 50%-60% normal hsp70 levels. These data demonstrate that TFIIS is required in vivo for maximal expression of hsp70 and suggest that TFIIS may serve to regulate the kinetics of the heat shock response by maintaining Pol II in a readily inducible conformation (Adelman, 2005).

These results suggest that TFIIS is involved in mediating the magnitude and efficiency of the heat shock response; additionally, TFIIS has been proposed to function broadly in transcription elongation by Pol II. To view the distribution of TFIIS both over the entire genome and at heat shock loci, Drosophila polytene chromosomes were stained with an antibody that is highly specific for TFIIS. Over 150 specific loci are stained by anti-TFIIS, including several interbands and chromosomal puffs, which contain the Pol II-transcribed developmental genes, the native and transgenic heat shock genes, and the nucleolus organizer, which contains the Pol I-transcribed rRNA genes. The consistent, prominent labeling of the nucleolus organizer suggests that TFIIS plays a role in Pol I elongation. A functional interaction between TFIIS and Pol I has been reported previously; however, conflicting reports have indicated that Pol I transcription is stimulated by a distinct transcript cleavage factor (Adelman, 2005).

Perhaps most surprising is the strong staining of many condensed chromosomal bands. These are sites that are not actively transcribed by RNA Pol I, II, or III. These transcriptionally inactive regions of TFIIS accumulation may be indicative of an as yet uncharacterized function of TFIIS, or may represent storage or proposed transcriptosome assembly loci akin to the TFIIS-containing Cajal Bodies in Xenopus oocytes (Adelman, 2005).

Upon stimulation of the heat shock response, TFIIS accumulates at heat shock loci. However, in contrast to many other transcription factors, TFIIS can still be observed at many other loci on the chromosomes, and in particular, the strong colocalization with condensed DNA bands persists. This result is consistent with recent data on the localization of TFIIS in yeast (Pokholok, 2002), where it was noted that TFIIS was not generally required for Pol II transcription but appeared to be specifically recruited to actively transcribed genes during times of cellular stress and when transcription was compromised (i.e., 6-AU treatment or temperature shift). In agreement with these results, Drosophila TFIIS is recruited to heat shock loci rapidly after heat induction and TFIIS appears to travel into the body of the gene along with Pol II, since it can be seen to colocalize throughout the puff with active Pol II (Adelman, 2005).

To analyze the localization of TFIIS at hsp70 at higher resolution, chromatin immunoprecipitation (ChIP) assays were performed followed by real-time PCR. This method allows for quantitative analysis of both the spatial and temporal distribution of TFIIS on the hsp70 gene. Pol II (detected using an antibody that recognizes the Pol II Rpb3 subunit) is associated specifically with the promoter region of hsp70 prior to heat shock (Boehm, 2003). Upon heat induction, Pol II is rapidly detected in the body of the gene and a robust recruitment of additional Pol II is observed (Adelman, 2005).

Strikingly, TFIIS is also present at the uninduced hsp70 promoter. This result is consistent with the idea that TFIIS associates with the promoter-proximal stalled Pol II to rescue it from arrest, thereby maintaining the Pol II in a rapidly responsive, active state. During heat shock, TFIIS is further recruited to the promoter region of hsp70 and TFIIS is seen to track along with the elongating Pol II into the body of the gene, in agreement with its role as an accessory factor for transcription elongation (Adelman, 2005).

All of the above data are consistent with the hypothesis that the promoter-proximal stalled Pol II has a tendency to fall into transcription arrest and that TFIIS serves to rescue the arrested Pol II so that it can be induced to elongate upon heat shock. Thus, one would predict that, in the absence of TFIIS, Pol II that becomes inactive in the promoter-proximal region would remain inactive, thereby presenting a steric obstacle to the rapid recruitment of additional Pol II molecules upon heat shock. The Pol II density at the hsp70 promoter before heat shock would thus remain unchanged (i.e., one Pol II present within each hsp70 promoter region), but the movement of Pol II into the body of the gene and the recruitment of additional Pol II upon heat shock should be diminished or delayed (Adelman, 2005).

To test this idea, a protocol was developed to perform ChIP on S2 cells that had been depleted of TFIIS by RNAi. TFIIS and LacZ RNAi-treated cells were crosslinked directly, or after a short, 2.5 min, heat shock. Depletion of TFIIS has no effect on the level of Pol II detected in the hsp70 promoter region before heat shock. This result indicates that TFIIS is not required for Pol II to stall within the promoter-proximal region. However, depletion of TFIIS leads to a significant reduction in the heat shock-induced recruitment of Pol II to the promoter. In fact, the Pol II density remains equivalent to that observed before heat induction. Moreover, the reduction in recruitment of Pol II is accompanied by a decrease in the Pol II signal throughout the body of the gene. This result indicates that the stalled Pol II is not efficiently released into the gene in the TFIIS-depleted cells, and that this “stuck” Pol II blocks recruitment of additional Pol II (Adelman, 2005).

As a control for the level of depletion, the presence of TFIIS at hsp70 was assayed in LacZ and TFIIS-treated cells. The 10-fold depletion of TFIIS observed by Western analysis leads to a similar reduction in TFIIS detectable on the hsp70 gene under both NHS and HS conditions. Importantly, depletion of TFIIS has no effect on the levels of HSF recruited to hsp70 upon heat shock, indicating that TFIIS-depleted cells did not display a general, nonspecific loss of factor recruitment. These results demonstrate that, while TFIIS is not required to establish the stalled Pol II at hsp70, depletion of TFIIS interferes with efficient release of Pol II from the promoter region and the rapid recruitment of additional Pol II (Adelman, 2005).

Pol II and/or general transcription factors have been found to occupy a growing number of promoters of preactivated genes. These varied promoters may utilize similar mechanisms for selectively recruiting certain components of the transcription machinery and for regulating transcription initiation and elongation through the promoter-proximal region. The efficiency of synthesis through the initially transcribed sequence is particularly sensitive to perturbation and is thus a prime target for gene regulation. Factors that impede the progress of the RNA polymerase within the first 10–40 nt, which often include both protein components and the nucleic acid sequence, have been shown to influence transcriptional pausing, arrest, and termination efficiency. Identification and characterization of the factors that modulate the regulatory pausing and/or stalling of Pol II within the promoter-proximal region is essential to understanding the regulation of genes like hsp70, wherein this step is rate limiting for gene expression (Adelman, 2005).

Transcription of hsp70 in vitro revealed positions of pausing that corresponded faithfully with locations that had been identified in vivo as harboring Pol II complexes that were not efficiently elongated. Likewise, Pol II artificially halted at these positions in vitro rapidly lost the capacity to resume transcription, even after removal of negatively acting elongation factors through stringent washing with sarkosyl. These results are similar to in vitro data reported by Gilmour and colleagues (Li, 1996), demonstrating that Pol II can become inactive within the promoter-proximal region. This work expands upon these observations by establishing that the inactive Pol II can be rescued by transcript cleavage factor TFIIS and thus represent arrested species. It is interesting to note that the predominant sites at which Pol II is found on the uninduced hsp70 gene (Rasmussen, 1993; Rasmussen, 1995) are positions to which Pol II stably backtracks in vitro (Adelman, 2005).

These data suggest the following model for the role of TFIIS in hsp70 gene expression. Under uninduced conditions, Pol II is recruited to the hsp70 promoter and begins to transcribe through the promoter-proximal region. Intrinsic pause sites within the initially transcribed sequence induce transient stops in elongation, giving the regulatory negative-elongation factors time to bind and impede further movement into the gene. However, Pol II stalled for an extended time at the pausing sites have a tendency to backtrack along the template, displacing the 3′ end of the RNA from the catalytic site and prohibiting further elongation. In the absence of TFIIS, the arrested, inactive Pol II are unable to resume transcription rapidly upon heat induction, even after the negatively acting factors have been removed. However, in the presence of TFIIS, TFIIS-dependent cleavage returns inactive Pol II to a transcriptionally active conformation so that, upon heat shock and the removal of negatively acting factors, Pol II can be rapidly released from the promoter region. The movement of the first Pol II away from the promoter region allows for the recruitment of subsequent Pol II molecules. It is noted that this model is supported by a recent study of factors that interact genetically with Dst1 (the yeast gene encoding TFIIS), which suggested a general role for TFIIS in the transition from initiation to elongation (Malagon, 2004; Adelman, 2005 and references therein).

These results are reminiscent of the role of bacterial Gre factors in mediating transcription efficiency through a regulatory pause in the late gene operon of λ bacteriophage (Marr, 2000). In the λ system, interactions between the RNA polymerase σ subunit and the promoter-proximal DNA sequence induce a transient pause in transcription, during which the λ Q protein binds and modifies the RNA polymerase, rendering it termination resistant. The Gre proteins modulate the kinetics of transcription through the pause site and are required for efficient function of the λ Q protein. Similarly, the activity of transcript cleavage factor TFIIS is necessary for efficient induction of hsp70 through its activation of promoter-proximally stalled Pol II. Thus, the current results indicate that, in addition to structural and mechanistic similarity between the Gre and TFIIS proteins, these factors may perform similar roles in vivo, serving to mediate the expression of genes that undergo pausing within the initially transcribed sequence (Adelman, 2005).

Search PubMed for articles about Drosophila TFIIS

Adelman, K., et al. (2005). Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol. Cell 17: 103-112. PubMed ID: 15629721

Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T. (2003). Transcription factor and polymerase recruitment, modification, and movement on dhsp70 in vivo in the minutes following heat shock. Mol Cell Biol. 23(21): 7628-37. PubMed ID: 14560008

Borukhov, S., Sagitov, V. and Goldfarb, A. (1993). Transcript cleavage factors from E. coli. Cell 72: 459-466. PubMed ID: 8431948

Fish, R. N. and Kane, C. M. (2002). Promoting elongation with transcript cleavage stimulatory factors. Biochim. Biophys. Acta 1577: 287-307. PubMed ID: 12213659

Kettenberger, H., Armache, K. J. and Cramer, P. (2003). Architecture of the RNA polymerase II-TFIIS complex and implications for mRNA cleavage. Cell 114: 347-357. PubMed ID: 12914699

Komissarova, N. and Kashlev, M. (1997). Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded. Proc. Natl. Acad. Sci. 94: 1755-1760. PubMed ID: 9050851

Laptenko, O., Lee, J., Lomakin, I. and Borukhov, S. (2003). Transcript cleavage factors GreA and GreB act as transient catalytic components of RNA polymerase. EMBO J. 22: 6322-6334. PubMed ID: 14633991

Li, B., Weber, J. A., Chen, Y., Greenleaf, A. L. and Gilmour, D. S. (1996). Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract. Mol. Cell. Biol. 16: 5433-5443. PubMed ID: 8816456

Marr, M. T. and Roberts, J. W. (2000). Function of transcription cleavage factors GreA and GreB at a regulatory pause site. Mol. Cell 6: 1275-1285. PubMed ID: 11163202

Opalka, N., Chlenov, M., Chacon, P., Rice, W. J., Wriggers, W. and Darst, S. A. (2003). Structure and function of the transcription elongation factor GreB bound to bacterial RNA polymerase. Cell 114: 335-345. PubMed ID: 12914698

Pokholok, D. K., Hannett, N. M. and Young, R. A. (2002). Exchange of RNA polymerase II initiation and elongation factors during gene expression in vivo. Mol. Cell 9: 799-809. PubMed ID: 11983171

Rasmussen, E. B. and Lis, J. T. (1993). In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes. Proc. Natl. Acad. Sci. 90: 7923-7927. PubMed ID: 8367444

Rasmussen, E. B. and Lis, J. T. (1995). Short transcripts of the ternary complex provide insight into RNA polymerase II elongational pausing. J. Mol. Biol. 252: 522-535. PubMed ID: 7563071

Sosunova, E., Sosunov, V., Kozlov, M., Nikiforov, V., Goldfarb, A. and Mustaev, A. (2003). Donation of catalytic residues to RNA polymerase active center by transcription factor Gre. Proc. Natl. Acad. Sci. 100: 15469-15474. PubMed ID: 14668436

Wind, M. and Reines, D. (2000). Transcription elongation factor SII. Bioessays 22: 327-336. PubMed ID: 10723030

date revised: 30 November 2007

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