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. 1997 Oct 15;25(20):4079–4084. doi: 10.1093/nar/25.20.4079

The RNA polymerase II preinitiation complex formed in the presence of ATP.

H Serizawa 1, Z Tsuchihashi 1, K Mizumoto 1
PMCID: PMC147005  PMID: 9321661

Abstract

ATP hydrolysis is required for transcriptional initiation by RNA polymerase II in vitro. Reconstituted transcription using purified initiation factors and RNA polymerase II have revealed that the step dependent on ATP hydrolysis occurs at the same time as initiation of RNA synthesis. We report here that ATP hydrolysis is also required for formation of the preinitiation complex in crude extracts. Two distinct preinitiation complexes were identified, one formed in the presence and the other in the absence of ATP. These complexes were isolated by glycerol gradient centrifugation. The preinitiation complex formed in the presence of ATP was able to synthesize transcripts with addition of only ribonucleotide triphosphates, whereas the preinitiation complex formed in the absence of ATP was inactive and required addition of protein fractions and ATP. These results suggest that the inactive preinitiation complex is activated by addition of the protein fractions and ATP hydrolysis. The active preinitiation complex sedimented at approximately 40 S in glycerol gradient centrifugation, a rate similar to that of RNA polymerase II holoenzyme reported by Maldonado et al. [ Nature (1996), 381, 86-89].

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Selected References

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  1. Björklund S., Kim Y. J. Mediator of transcriptional regulation. Trends Biochem Sci. 1996 Sep;21(9):335–337. doi: 10.1016/s0968-0004(96)10051-7. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Bunick D., Zandomeni R., Ackerman S., Weinmann R. Mechanism of RNA polymerase II--specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts. Cell. 1982 Jul;29(3):877–886. doi: 10.1016/0092-8674(82)90449-4. [DOI] [PubMed] [Google Scholar]
  4. Buratowski S., Hahn S., Guarente L., Sharp P. A. Five intermediate complexes in transcription initiation by RNA polymerase II. Cell. 1989 Feb 24;56(4):549–561. doi: 10.1016/0092-8674(89)90578-3. [DOI] [PubMed] [Google Scholar]
  5. Chao D. M., Gadbois E. L., Murray P. J., Anderson S. F., Sonu M. S., Parvin J. D., Young R. A. A mammalian SRB protein associated with an RNA polymerase II holoenzyme. Nature. 1996 Mar 7;380(6569):82–85. doi: 10.1038/380082a0. [DOI] [PubMed] [Google Scholar]
  6. Conaway R. C., Conaway J. W. ATP activates transcription initiation from promoters by RNA polymerase II in a reversible step prior to RNA synthesis. J Biol Chem. 1988 Feb 25;263(6):2962–2968. [PubMed] [Google Scholar]
  7. Conaway R. C., Conaway J. W. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7356–7360. doi: 10.1073/pnas.86.19.7356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conaway R. C., Conaway J. W. General initiation factors for RNA polymerase II. Annu Rev Biochem. 1993;62:161–190. doi: 10.1146/annurev.bi.62.070193.001113. [DOI] [PubMed] [Google Scholar]
  9. Conaway R. C., Garrett K. P., Hanley J. P., Conaway J. W. Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors alpha and beta gamma promote entry of polymerase into the preinitiation complex. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6205–6209. doi: 10.1073/pnas.88.14.6205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drapkin R., Reardon J. T., Ansari A., Huang J. C., Zawel L., Ahn K., Sancar A., Reinberg D. Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature. 1994 Apr 21;368(6473):769–772. doi: 10.1038/368769a0. [DOI] [PubMed] [Google Scholar]
  12. Dvir A., Garrett K. P., Chalut C., Egly J. M., Conaway J. W., Conaway R. C. A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation. J Biol Chem. 1996 Mar 29;271(13):7245–7248. doi: 10.1074/jbc.271.13.7245. [DOI] [PubMed] [Google Scholar]
  13. Feaver W. J., Gileadi O., Li Y., Kornberg R. D. CTD kinase associated with yeast RNA polymerase II initiation factor b. Cell. 1991 Dec 20;67(6):1223–1230. doi: 10.1016/0092-8674(91)90298-d. [DOI] [PubMed] [Google Scholar]
  14. Feaver W. J., Svejstrup J. Q., Bardwell L., Bardwell A. J., Buratowski S., Gulyas K. D., Donahue T. F., Friedberg E. C., Kornberg R. D. Dual roles of a multiprotein complex from S. cerevisiae in transcription and DNA repair. Cell. 1993 Dec 31;75(7):1379–1387. doi: 10.1016/0092-8674(93)90624-y. [DOI] [PubMed] [Google Scholar]
  15. Feaver W. J., Svejstrup J. Q., Henry N. L., Kornberg R. D. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK. Cell. 1994 Dec 16;79(6):1103–1109. doi: 10.1016/0092-8674(94)90040-x. [DOI] [PubMed] [Google Scholar]
  16. Flores O., Lu H., Killeen M., Greenblatt J., Burton Z. F., Reinberg D. The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9999–10003. doi: 10.1073/pnas.88.22.9999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holstege F. C., van der Vliet P. C., Timmers H. T. Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH. EMBO J. 1996 Apr 1;15(7):1666–1677. [PMC free article] [PubMed] [Google Scholar]
  18. Imbalzano A. N., Kwon H., Green M. R., Kingston R. E. Facilitated binding of TATA-binding protein to nucleosomal DNA. Nature. 1994 Aug 11;370(6489):481–485. doi: 10.1038/370481a0. [DOI] [PubMed] [Google Scholar]
  19. Jiang Y., Yan M., Gralla J. D. Abortive initiation and first bond formation at an activated adenovirus E4 promoter. J Biol Chem. 1995 Nov 10;270(45):27332–27338. doi: 10.1074/jbc.270.45.27332. [DOI] [PubMed] [Google Scholar]
  20. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  21. Koleske A. J., Young R. A. An RNA polymerase II holoenzyme responsive to activators. Nature. 1994 Mar 31;368(6470):466–469. doi: 10.1038/368466a0. [DOI] [PubMed] [Google Scholar]
  22. Koleske A. J., Young R. A. The RNA polymerase II holoenzyme and its implications for gene regulation. Trends Biochem Sci. 1995 Mar;20(3):113–116. doi: 10.1016/s0968-0004(00)88977-x. [DOI] [PubMed] [Google Scholar]
  23. Kwon H., Imbalzano A. N., Khavari P. A., Kingston R. E., Green M. R. Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex. Nature. 1994 Aug 11;370(6489):477–481. doi: 10.1038/370477a0. [DOI] [PubMed] [Google Scholar]
  24. Li Y., Kornberg R. D. Interplay of positive and negative effectors in function of the C-terminal repeat domain of RNA polymerase II. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2362–2366. doi: 10.1073/pnas.91.6.2362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lu H., Zawel L., Fisher L., Egly J. M., Reinberg D. Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II. Nature. 1992 Aug 20;358(6388):641–645. doi: 10.1038/358641a0. [DOI] [PubMed] [Google Scholar]
  26. Luse D. S., Jacob G. A. Abortive initiation by RNA polymerase II in vitro at the adenovirus 2 major late promoter. J Biol Chem. 1987 Nov 5;262(31):14990–14997. [PubMed] [Google Scholar]
  27. Maldonado E., Shiekhattar R., Sheldon M., Cho H., Drapkin R., Rickert P., Lees E., Anderson C. W., Linn S., Reinberg D. A human RNA polymerase II complex associated with SRB and DNA-repair proteins. Nature. 1996 May 2;381(6577):86–89. doi: 10.1038/381086a0. [DOI] [PubMed] [Google Scholar]
  28. Manley J. L., Fire A., Cano A., Sharp P. A., Gefter M. L. DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3855–3859. doi: 10.1073/pnas.77.7.3855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mäkelä T. P., Parvin J. D., Kim J., Huber L. J., Sharp P. A., Weinberg R. A. A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription. Proc Natl Acad Sci U S A. 1995 May 23;92(11):5174–5178. doi: 10.1073/pnas.92.11.5174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rappaport J., Weinmann R. Purine triphosphate beta-gamma bond hydrolysis requirements for RNA polymerase II transcription initiation and elongation. J Biol Chem. 1987 Dec 25;262(36):17510–17515. [PubMed] [Google Scholar]
  31. Reinberg D., Roeder R. G. Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE. J Biol Chem. 1987 Mar 5;262(7):3310–3321. [PubMed] [Google Scholar]
  32. Roy R., Adamczewski J. P., Seroz T., Vermeulen W., Tassan J. P., Schaeffer L., Nigg E. A., Hoeijmakers J. H., Egly J. M. The MO15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor. Cell. 1994 Dec 16;79(6):1093–1101. doi: 10.1016/0092-8674(94)90039-6. [DOI] [PubMed] [Google Scholar]
  33. Safer B., Yang L., Tolunay H. E., Anderson W. F. Isolation of stable preinitiation, initiation, and elongation complexes from RNA polymerase II-directed transcription. Proc Natl Acad Sci U S A. 1985 May;82(9):2632–2636. doi: 10.1073/pnas.82.9.2632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sawadogo M., Roeder R. G. Energy requirement for specific transcription initiation by the human RNA polymerase II system. J Biol Chem. 1984 Apr 25;259(8):5321–5326. [PubMed] [Google Scholar]
  35. Schaeffer L., Moncollin V., Roy R., Staub A., Mezzina M., Sarasin A., Weeda G., Hoeijmakers J. H., Egly J. M. The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor. EMBO J. 1994 May 15;13(10):2388–2392. doi: 10.1002/j.1460-2075.1994.tb06522.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermeulen W., Hoeijmakers J. H., Chambon P., Egly J. M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science. 1993 Apr 2;260(5104):58–63. doi: 10.1126/science.8465201. [DOI] [PubMed] [Google Scholar]
  37. Serizawa H., Conaway J. W., Conaway R. C. Phosphorylation of C-terminal domain of RNA polymerase II is not required in basal transcription. Nature. 1993 May 27;363(6427):371–374. doi: 10.1038/363371a0. [DOI] [PubMed] [Google Scholar]
  38. Serizawa H., Conaway R. C., Conaway J. W. A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7476–7480. doi: 10.1073/pnas.89.16.7476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Serizawa H., Conaway R. C., Conaway J. W. Multifunctional RNA polymerase II initiation factor delta from rat liver. Relationship between carboxyl-terminal domain kinase, ATPase, and DNA helicase activities. J Biol Chem. 1993 Aug 15;268(23):17300–17308. [PubMed] [Google Scholar]
  40. Serizawa H., Mäkelä T. P., Conaway J. W., Conaway R. C., Weinberg R. A., Young R. A. Association of Cdk-activating kinase subunits with transcription factor TFIIH. Nature. 1995 Mar 16;374(6519):280–282. doi: 10.1038/374280a0. [DOI] [PubMed] [Google Scholar]
  41. Shiekhattar R., Mermelstein F., Fisher R. P., Drapkin R., Dynlacht B., Wessling H. C., Morgan D. O., Reinberg D. Cdk-activating kinase complex is a component of human transcription factor TFIIH. Nature. 1995 Mar 16;374(6519):283–287. doi: 10.1038/374283a0. [DOI] [PubMed] [Google Scholar]
  42. Uetsuki T., Naito A., Nagata S., Kaziro Y. Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor-1 alpha. J Biol Chem. 1989 Apr 5;264(10):5791–5798. [PubMed] [Google Scholar]
  43. Weil P. A., Luse D. S., Segall J., Roeder R. G. Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA. Cell. 1979 Oct;18(2):469–484. doi: 10.1016/0092-8674(79)90065-5. [DOI] [PubMed] [Google Scholar]
  44. Wilson C. J., Chao D. M., Imbalzano A. N., Schnitzler G. R., Kingston R. E., Young R. A. RNA polymerase II holoenzyme contains SWI/SNF regulators involved in chromatin remodeling. Cell. 1996 Jan 26;84(2):235–244. doi: 10.1016/s0092-8674(00)80978-2. [DOI] [PubMed] [Google Scholar]
  45. Zawel L., Reinberg D. Initiation of transcription by RNA polymerase II: a multi-step process. Prog Nucleic Acid Res Mol Biol. 1993;44:67–108. doi: 10.1016/s0079-6603(08)60217-2. [DOI] [PubMed] [Google Scholar]

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