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• W2018092284 abstract "The positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II. The positive transcription elongation factor, P-TEFb, controls the fraction of initiated RNA polymerase II molecules that enter into the productive mode of elongation necessary to generate mRNAs. To better understand the mechanism of this transition into productive elongation we optimized a defined in vitro transcription system and compared results obtained with it to those obtained with a crude system. We found that controlling the function of TFIIF is a key aspect of RNA polymerase II elongation control. Before P-TEFb function, early elongation complexes under the control of negative factors are completely unresponsive to the robust elongation stimulatory activity of TFIIF. P-TEFb-mediated phosphorylation events, targeting the elongation complex containing DSIF and NELF, reverse the negative effect of DSIF and NELF and simultaneously facilitate the action of TFIIF. We also found that productive elongation complexes are completely resistant to negative elongation factors. Our data suggest that an additional factor(s) is involved in establishing the unique resistance activities of the elongation complexes before and after P-TEFb function. Furthermore, we provide evidence for the existence of another positive activity required for efficient function of P-TEFb. A model of the mechanism of P-TEFb-mediated elongation control is proposed in which P-TEFb induces the transition into productive elongation by changing the accessibility of elongation factors to elongation complexes. Our results have uncovered important properties of elongation complexes that allow a more complete understanding of how P-TEFb controls the elongation phases of transcription by RNA polymerase II. The elongation stage of eukaryotic RNA polymerase II (RNAPII) 2The abbreviations used are: RNAPII, RNA polymerase II; DSIF, DRB sensitivity-inducing factor; NELF, negative elongation factor; nt, nucleotide; EEC, early elongation complex; HNE, HeLa nuclear extract; ssDNA, single-stranded DNA; PEC, productive elongation complex; DRB, 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole; P-TEFb, positive transcription elongation factor b; AEC, abortive elongation complexes; CTD, C-terminal domain. transcription is not only essential for generating full-length mRNA but also is a critical target for the regulation of gene expression (1Bentley D.L. Curr. Opin. Genet. Dev. 1995; 5: 210-216Crossref PubMed Scopus (105) Google Scholar, 2Peterlin B.M. Price D.H. Mol. Cell. 2006; 23: 297-305Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). An elongation control process was initially uncovered during studies of the transcription inhibitory mechanism of the ATP analog DRB (3Sehgal P.B. Darnell Jr., J.E. Tamm I. Cell. 1976; 9: 473-480Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 4Tamm I. Kikuchi T. Darnell Jr., J.E. Salditt-Georgieff M. Biochemistry. 1980; 19: 2743-2748Crossref PubMed Scopus (41) Google Scholar). DRB treatment blocks RNAPII transcription specifically at an early step in elongation without inhibiting the enzymatic activity of purified RNAPII itself (4Tamm I. Kikuchi T. Darnell Jr., J.E. Salditt-Georgieff M. Biochemistry. 1980; 19: 2743-2748Crossref PubMed Scopus (41) Google Scholar, 5Marshall N.F. Price D.H. Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (242) Google Scholar). Further studies uncovered a new class of elongation factors responsible for this DRB sensitivity. Two negative elongation factors, the DRB sensitivity-inducing factor (DSIF) and the negative elongation factor (NELF) cause transcription pausing by physically associating with RNAPII (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar, 7Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 8Zhang Z. Wu C.H. Gilmour D.S. J. Biol. Chem. 2004; 279: 23223-23228Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Positive transcription elongation factor b (P-TEFb), a cyclindependent kinase that can be inhibited by DRB, counteracts the negative effects of DSIF and NELF and allows RNAPII to enter productive elongation (9Marshall N.F. Price D.H. J. Biol. Chem. 1995; 270: 12335-12338Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, 10Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 12Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (613) Google Scholar, 13Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1055) Google Scholar, 14Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (282) Google Scholar). Early studies identified many genes that were regulated at the stage of transcription elongation, including hsp70 (15Rougvie A.E. Lis J.T. Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (469) Google Scholar), c-myc (16Krumm A. Meulia T. Brunvand M. Groudine M. Genes Dev. 1992; 6: 2201-2213Crossref PubMed Scopus (221) Google Scholar, 17Roberts S. Purton T. Bentley D.L. Genes Dev. 1992; 6: 1562-1574Crossref PubMed Scopus (17) Google Scholar), and the HIV-LTR (18Jones K.A. Peterlin B.M. Annu. Rev. Biochem. 1994; 63: 717-743Crossref PubMed Scopus (559) Google Scholar), and later a common regulatory mechanism utilized by these genes was uncovered, which was the recruitment of P-TEFb to the transcription machinery (12Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (613) Google Scholar, 19Lis J.T. Mason P. Peng J. Price D.H. Werner J. Genes Dev. 2000; 14: 792-803PubMed Google Scholar, 20Majello B. Napolitano G. Giordano A. Lania L. Oncogene. 1999; 18: 4598-4605Crossref PubMed Scopus (59) Google Scholar). Indeed, P-TEFb is the key regulator that causes RNAPII to overcome the rate-limiting step during the early stage of elongation. P-TEFb is an essential cellular coactivator for the viral transactivator Tat in stimulating transcription from HIV-LTR (12Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (613) Google Scholar). Accumulating evidence indicates that control of gene expression by P-TEFb plays an important role in cellular activation, proliferation, and differentiation (2Peterlin B.M. Price D.H. Mol. Cell. 2006; 23: 297-305Abstract Full Text Full Text PDF PubMed Scopus (853) Google Scholar). It has been shown that many causes of cardiac hypertrophy converge at the critical step of up-regulation of P-TEFb activity (21Kulkarni P.A. Sano M. Schneider M.D. Rec. Progr. Hormone Res. 2004; 59: 125-139Crossref PubMed Scopus (20) Google Scholar, 22Sano M. Abdellatif M. Oh H. Xie M. Bagella L. Giordano A. Michael L.H. DeMayo F.J. Schneider M.D. Nat. Med. 2002; 8: 1310-1317Crossref PubMed Scopus (204) Google Scholar). High P-TEFb activity may also play a role in maintenance of the cancer state, evidenced by the fact that one potential anti-cancer drug, flavopiridol, has been found to act as a potent P-TEFb inhibitor (23Chao S.H. Price D.H. J. Biol. Chem. 2001; 276: 31793-31799Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Although required for the productive elongation of many genes, P-TEFb by itself has been shown to have no direct effect on RNAPII elongation rate in vitro, emphasizing that P-TEFb functions as a regulator of other elongation factors (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). P-TEFb can phosphorylate several proteins during elongation. It is responsible for hyperphosphorylation of the serine 2 positions of heptapeptide repeats in the C-terminal domain (CTD) of the large subunit of RNAPII (10Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Similarly, the CTD-like heptapeptide repeats in the C-terminal region of the hSpt5 subunit of DSIF can also be phosphorylated by P-TEFb, which is crucial for the positive elongation activity of DSIF (24Yamada T. Yamaguchi Y. Inukai N. Okamoto S. Mura T. Handa H. Mol. Cell. 2006; 21: 227-237Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). In addition, phosphorylation of one of the NELF subunits, NELFe, has been correlated with dissociation of NELF from elongation complexes that contain nascent RNA with double-stranded regions (25Fujinaga K. Irwin D. Huang Y. Taube R. Kurosu T. Peterlin B.M. Mol. Cell. Biol. 2004; 24: 787-795Crossref PubMed Scopus (261) Google Scholar). It remains unclear if phosphorylation of all or only a subset of these P-TEFb substrates is directly responsible and sufficient for the regulation of elongation. Indeed, P-TEFb-mediated phosphorylations on RNAPII-CTD and DSIF are known to be involved in pre-mRNA processing (26Ni Z. Schwartz B.E. Werner J. Suarez J.R. Lis J.T. Mol. Cell. 2004; 13: 55-65Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 27Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7572-7577Crossref PubMed Scopus (135) Google Scholar, 28Bird G. Zorio D.A. Bentley D.L. Mol. Cell. Biol. 2004; 24: 8963-8969Crossref PubMed Scopus (96) Google Scholar, 29Lindstrom D.L. Squazzo S.L. Muster N. Burckin T.A. Wachter K.C. Emigh C.A. McCleery J.A. Yates 3rd, J.R. Hartzog G.A. Mol. Cell. Biol. 2003; 23: 1368-1378Crossref PubMed Scopus (212) Google Scholar). Moreover, exactly how P-TEFb-mediated phosphorylations cause functional changes in elongation complexes is not well understood. Other known elongation factors such as TFIIF (30Price D.H. Sluder A.E. Greenleaf A.L. Mol. Cell. Biol. 1989; 9: 1465-1475Crossref PubMed Scopus (134) Google Scholar), TFIIS (31Guo H. Price D.H. J. Biol. Chem. 1993; 268: 18762-18770Abstract Full Text PDF PubMed Google Scholar), and elongin (32Aso T. Lane W.S. Conaway J.W. Conaway R.C. Science (New York, NY). 1995; 269: 1439-1443Crossref Scopus (293) Google Scholar) affect elongation, but their role in the elongation control process is unclear (33Reines D. Conaway J.W. Conaway R.C. Trends Biochem. Sci. 1996; 21: 351-355Abstract Full Text PDF PubMed Scopus (120) Google Scholar). DSIF has been implicated as both a positive and negative elongation factor (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar). In humans it is composed of two subunits (160 and 14 kDa) that are homologs of yeast proteins Spt5 and Spt4 (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar). Its transcription repression activity requires NELF and can be alleviated by P-TEFb (7Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, 14Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (282) Google Scholar). Therefore, it has been proposed that P-TEFb causes the transition of RNAPII into productive elongation mode by reversing the negative effect of DSIF and NELF (14Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (282) Google Scholar, 34Price D.H. Mol. Cell. Biol. 2000; 20: 2629-2634Crossref PubMed Scopus (571) Google Scholar). Recent studies suggested that phosphorylation of DSIF by P-TEFb can switch it into a positive elongation factor (24Yamada T. Yamaguchi Y. Inukai N. Okamoto S. Mura T. Handa H. Mol. Cell. 2006; 21: 227-237Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). DSIF has been shown to have a stimulatory effect on elongation only in reactions containing nuclear extract and limiting concentrations of ribonucleoside triphosphates (6Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (571) Google Scholar), but it has no effects in the absence of other proteins (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The exact role of DSIF and other factors in stimulating the rate of transcription after P-TEFb function and the mechanisms utilized to transduce the signal from P-TEFb are not known. Most of the factors affecting RNAPII elongation were identified through utilizing functional assays that provided a means of purifying factors that had observable effects on the native elongation properties of RNAPII (35Adamson T.E. Shore S.M. Price D.H. Methods Enzymol. 2003; 371: 264-275Crossref PubMed Scopus (24) Google Scholar). Previously, we developed an in vitro transcription system using an immobilized DNA template to study elongation control of RNAPII (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 35Adamson T.E. Shore S.M. Price D.H. Methods Enzymol. 2003; 371: 264-275Crossref PubMed Scopus (24) Google Scholar). Early elongation complexes containing pulse-labeled nascent RNAs were isolated and allowed to further extend transcripts in the presence of elongation factors that were added back. When purified DSIF and NELF along with recombinant P-TEFb were tested, we confirmed that the two negative factors, when present together, were able to slow the elongation of RNAPII and this effect could be eliminated by P-TEFb (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Our data also demonstrated that the general transcription factor, TFIIF could dramatically stimulate the elongation rate and could functionally compete with DSIF and NELF in controlling RNAPII elongation (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In this study, we employed an in vitro transcription system optimized to further dissect the functional mechanisms of P-TEFb-mediated elongation control. We found that P-TEFb-directed phosphorylation events can be fulfilled without active transcription, making it possible to more thoroughly study the functional targets of P-TEFb. During the course of our studies, we realized that the properties of elongation complexes are completely resistant to known positive factors before P-TEFb function and completely resistant to negative factors after P-TEFb function. In addition, our data strongly suggest that P-TEFb not only reverses the negative effect of DSIF and NELF, but also facilitates the function of TFIIF as the major positive elongation factor in productive elongation. Materials—HeLa nuclear extract (HNE) was prepared as described by Adamson et al. (35Adamson T.E. Shore S.M. Price D.H. Methods Enzymol. 2003; 371: 264-275Crossref PubMed Scopus (24) Google Scholar). Bacterially expressed human DSIF was purified as described by Renner et al. (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). NELF was affinity-purified from HeLa S3 cells stably transfected with FLAG-tagged NELFe as described by Renner et al. (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). P-TEFb containing Cdk9 and cyclin T2a was expressed in baculovirus-infected insect cells and purified as described by Peng et al. (36Peng J. Zhu Y. Milton J.T. Price D.H. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (452) Google Scholar). Recombinant human TFIIF was purified as described in Peng et al. (37Peng J. Liu M. Marion J. Zhu Y. Price D.H. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 365-370Crossref PubMed Scopus (31) Google Scholar). Isolation of Early Elongation Complexes (EECs)—An immobilized DNA template was generated as previously described which contained the full cytomegalovirus promoter driving the production of a 548 nt run-off transcript (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The protocol used to generate early elongation complexes was also as previously described (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 35Adamson T.E. Shore S.M. Price D.H. Methods Enzymol. 2003; 371: 264-275Crossref PubMed Scopus (24) Google Scholar) except for some modifications. For each individual transcription reaction, 8 μl of preincubation mixture containing 200 ng of template (∼0.5 pmol) and 1 μl of HNE was incubated with 20 mm HEPES, 60 mm KCl, 7 mm MgCl2, 10 units of RNaseOUT™ (Invitrogen), and 1 μm flavopiridol for 10 min at room temperature. Transcription was initiated upon the addition of physiological concentrations of ATP, GTP, UTP (500 μm), and 5 μCi of [α-32P]CTP. After 30 s of pulse, elongation was halted with the addition of EDTA to 20 mm, and the resultant EECs contained labeled nascent RNA predominantly less than 25 nt in length. Complexes associated with the immobilized templates were stringently washed three times with high salt EEC isolation buffer (20 mm HEPES and 1.6 m KCl) followed by two washes with low salt EEC isolation buffer (20 mm HEPES, 60 mm KCl, and 200 μg/ml bovine serum albumin), and resuspended in low salt EEC isolation buffer. The isolated EECs used in each experiment were generated in one large reaction, isolated, and aliquoted into individual elongation reactions. Transcript Extension (Chase)—Extension of transcripts from isolated EECs was carried out in 18-μl reactions by first mixing the isolated complexes with either HNE or purified factors in transcription buffer containing 20 mm HEPES, 60 mm KCl, 200 μg/ml bovine serum albumin, 3 mm MgCl2, and 10 units of RNaseOUT™. Elongation was then resumed upon the addition of NTPs to 500 μm and allowed to proceed for the indicated amounts of time at room temperature. Except for specific indications, we carried out the elongation reactions for 7 min. The reactions were stopped by the addition of 200 μl of Stop Solution (100 mm Tris, 100 mm NaCl, 10 mm EDTA, 1% Sarkosyl, 200 μg/ml Torula yeast RNA (Sigma)). RNA preparation and analyses on denaturing gels were described previously (38Price D.H. Sluder A.E. Greenleaf A.L. J. Biol. Chem. 1987; 262: 3244-3255Abstract Full Text PDF PubMed Google Scholar). Autoradiography of the dried gels provided images of the results, and quantitation was accomplished with a Packard InstantImager (PerkinElmer Life Sciences). Prephosphorylation Reactions—Recombinant P-TEFb and the indicated combinations of components, including preterminated EECs (see pretermination reactions below), DSIF, NELF, and HNE, were assembled in 9-μl reactions (half size of the final transcription elongation reactions) in the same transcript extension conditions as described above excluding NTPs. The prephosphorylation reactions were started upon the addition of ATP to 500 μm and were allowed to proceed for 5 min at room temperature, and then P-TEFb activity was inhibited by the addition of flavopiridol to 1 μm. When only a subset of the components was subjected to prephosphorylation, the indicated unphosphorylated components were supplemented after the termination of P-TEFb-directed prephosphorylation. Each of the final transcription reactions was 18 μl with the transcript extension conditions kept the same as above. Elongation was allowed to proceed for indicated times upon the addition of CTP, UTP, and GTP to 500 μm. Pre-termination Reactions—When isolated EECs were incubated with P-TEFb in the presence of 500 μm ATP, some polymerases terminated because of the presence of a trace amount of TTF2 remaining bound to the DNA template. TTF2 is a transcription termination factor and its dsDNA-dependent ATPase activity is required for releasing transcripts from the template (39Xie Z. Price D. J. Biol. Chem. 1997; 272: 31902-31907Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In the presence of ATP, TTF2 preferentially binds with ssDNA, and its ATPase activity is thus suppressed (40Xie Z. Price D.H. J. Biol. Chem. 1998; 273: 3771-3777Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). To limit the impact of contaminating TTF2 during prephosphorylation reactions and subsequent transcription reactions, we carried out a “pre-termination” reaction to inhibit the continuous activity of TTF2. Isolated EECs were incubated with 500 μm ATP, 3 mm MgCl2, and 100 ng per reaction of ssDNA (a 50-nt DNA oligo with random sequences, synthesized by IDT) at room temperature for 5 min. During this process, only a small fraction of EECs terminated, and once TTF2 came off from these terminated DNA it bound to ssDNA. The pre-terminated EECs were then washed with high salt EEC isolation buffer to remove the terminated polymerases, released RNA, contaminating TTF2 and ssDNA. Eventually, the beads were washed and resuspended with low salt EEC isolation buffer and ready for the prephosphorylation treatment. Amounts of HeLa Nuclear Extract or Purified Factors Used in Transcription Reactions—1 μl per reaction of HNE was used for generating early elongation complexes. The same amount of HNE was also used in indicated add-back assays except for specific indications. The amounts of recombinant DSIF and NELF were optimized to achieve the maximal negative effect on elongation and called “1×.” 1× of DSIF was 0.45 pmol per reaction and 1× of NELF was 0.03 pmol per reaction. 1× of TFIIF was 0.2 pmol, which is the lowest level that achieved the maximal increase in elongation rate in a 2-min reaction. 1× of P-TEFb was 3.3 pmol. 1× of factors was applied in either prephosphorylation reactions or in add-back assays unless otherwise indicated. A previously developed in vitro transcription system using an immobilized DNA template (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 35Adamson T.E. Shore S.M. Price D.H. Methods Enzymol. 2003; 371: 264-275Crossref PubMed Scopus (24) Google Scholar) was further optimized and employed here to explore the details of RNAPII elongation control by P-TEFb. The core of this technique is to determine the influence of a crude extract or purified elongation factors on elongation of nascent transcripts in isolated early elongation complexes (EECs) using add-back assays. A 5′-biotinylated DNA template containing the CMV immediate early promoter is immobilized onto paramagnetic beads through a biotin-streptavidin linkage, which allows for the subsequent isolation of the early elongation complexes. Preinitiation complexes are formed on the promoter by incubation with HNE. Upon the addition of nucleotides including limiting [α-32P]CTP, RNAPII initiates and generates labeled, nascent transcripts that are 15-20 nt in length within 30 s. Polymerases are halted by addition of EDTA and repeatedly washed with very high salt buffer followed by low salt washes and are finally resuspended in transcription buffer without nucleotides. The isolated EECs are then supplemented with either a crude extract or purified elongation factors and allowed to extend nascent transcripts upon addition of nucleotides. The transcripts are extracted and analyzed on a denaturing RNA gel and the effects of the factors on elongation are evaluated from the resultant change in the sizes of the RNAs. For brevity, we shall refer to the transcription assays with a HeLa nuclear extract added back or with only purified factors added back as the “crude system” or “defined system,” respectively. The defined system is named not because every component is known to be 100% pure, but because the components are highly purified and have defined methods of isolation. Although DSIF and NELF have been demonstrated to be very pure (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), it is possible that other proteins besides RNA polymerase II subunits may be present in the isolated elongation complexes (41Ujvari A. Luse D.S. J. Biol. Chem. 2004; 279: 49773-49779Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). A systematic examination of the high salt EEC isolation conditions was carried out, and we found that raising the salt from 1 to 1.6 m (data not shown) and elimination of the Sarkosyl resulted in the isolated EECs that responded more efficiently to DSIF and NELF and other elongation factors such as TFIIF (supplemental Fig. S1). This eliminated a serious limitation in our early attempt to reconstitute elongation control in a defined system in which only about half of the isolated EECs responded to DSIF and NELF (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). We believe that Sarkosyl was not completely removed during the following low salt washes, and this negatively affected the function of the factors added back, presumably, by interfering with their interaction with EECs. Reconstitution of P-TEFb-mediated Elongation Control in Vitro—Using EECs isolated under the new optimized conditions, we first examined the effect of recombinant DSIF and affinity-purified NELF on RNAPII elongation by examining the change in the kinetics of elongation. Isolated EECs were allowed to elongate for indicated times (from 0.5 to 16 min) in the absence or presence of DSIF and NELF (Fig. 1A). With no factors added back, isolated EECs that initially contained transcripts predominantly less than 25 nt in length (Fig. 1A, lane 1) moved slowly down the template at a relatively constant rate, as evidenced by the increase in the average lengths of the accumulated transcripts with time (Fig. 1A, lanes 2-7). The native elongation rate under these experimental conditions was about 25 nt per minute, and no run-off transcripts (548 nt) were detected even after 16 min of elongation. With the addition of DSIF and NELF, a decrease in the length of transcripts was observed at all the time points tested (Fig. 1A, lanes 8-13). Consistent with our previous report (11Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. J. Biol. Chem. 2001; 276: 42601-42609Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), DSIF and NELF appeared to significantly increase the time that RNAPII elongation complexes spent at intrinsic pause sites. Inclusion of DSIF and NELF resulted in the reduction of the elongation rate for almost all polymerases to about 30% of their native rate (Fig. 1A, compare lanes 8-13 to 2-7). Next we attempted to reconstitute P-TEFb-mediated elongation control using both a defined system and a crude system. Isolated EECs were allowed to elongate for 7 min with or without additional factors. The nascent transcripts were extende" @default.
• W2018092284 created "2016-06-24" @default.
• W2018092284 creator A5033510715 @default.
• W2018092284 creator A5074762963 @default.
• W2018092284 date "2007-07-01" @default.
• W2018092284 modified "2023-10-16" @default.
• W2018092284 title "Properties of RNA Polymerase II Elongation Complexes Before and After the P-TEFb-mediated Transition into Productive Elongation" @default.
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