Abstract
We investigated the role of TFIIH in transcription by RNA polymerase II (pol II) in vivo by microinjection of antibodies against this factor into Xenopus oocytes. Five different antibodies directed against four subunits of TFIIH were tested for effects on transcription of coinjected human immunodeficiency virus type 2 and c-myc templates. Each of these antibodies severely reduced the efficiency of elongation through human immunodeficiency virus type 2 and c-myc terminator elements. In contrast, an anti-TFIIB antibody did not inhibit elongation. Anti-TFIIH antibodies also had a much smaller inhibitory effect on total transcription than did anti-TFIIB or anti-pol II large subunit. Three inhibitors of TFIIH kinase activity, H-7, H-8, and dichlororibofuranosylbenzimidazole (DRB), inhibited elongation similarly to anti-TFIIH antibodies. These results strongly suggest a role for TFIIH in the stimulation of transcriptional elongation in vivo.
Full Text
The Full Text of this article is available as a PDF (846.0 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bentley D. L., Groudine M. Sequence requirements for premature termination of transcription in the human c-myc gene. Cell. 1988 Apr 22;53(2):245–256. doi: 10.1016/0092-8674(88)90386-8. [DOI] [PubMed] [Google Scholar]
- Blau J., Xiao H., McCracken S., O'Hare P., Greenblatt J., Bentley D. Three functional classes of transcriptional activation domain. Mol Cell Biol. 1996 May;16(5):2044–2055. doi: 10.1128/mcb.16.5.2044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Boerrigter M. E., Dollé M. E., Martus H. J., Gossen J. A., Vijg J. Plasmid-based transgenic mouse model for studying in vivo mutations. Nature. 1995 Oct 19;377(6550):657–659. doi: 10.1038/377657a0. [DOI] [PubMed] [Google Scholar]
- Chodosh L. A., Fire A., Samuels M., Sharp P. A. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. J Biol Chem. 1989 Feb 5;264(4):2250–2257. [PubMed] [Google Scholar]
- Cismowski M. J., Laff G. M., Solomon M. J., Reed S. I. KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity. Mol Cell Biol. 1995 Jun;15(6):2983–2992. doi: 10.1128/mcb.15.6.2983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Devault A., Martinez A. M., Fesquet D., Labbé J. C., Morin N., Tassan J. P., Nigg E. A., Cavadore J. C., Dorée M. MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK. EMBO J. 1995 Oct 16;14(20):5027–5036. doi: 10.1002/j.1460-2075.1995.tb00185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Drapkin R., Reinberg D. The multifunctional TFIIH complex and transcriptional control. Trends Biochem Sci. 1994 Nov;19(11):504–508. doi: 10.1016/0968-0004(94)90139-2. [DOI] [PubMed] [Google Scholar]
- Emerman M., Guyader M., Montagnier L., Baltimore D., Muesing M. A. The specificity of the human immunodeficiency virus type 2 transactivator is different from that of human immunodeficiency virus type 1. EMBO J. 1987 Dec 1;6(12):3755–3760. doi: 10.1002/j.1460-2075.1987.tb02710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Fischer L., Gerard M., Chalut C., Lutz Y., Humbert S., Kanno M., Chambon P., Egly J. M. Cloning of the 62-kilodalton component of basic transcription factor BTF2. Science. 1992 Sep 4;257(5075):1392–1395. doi: 10.1126/science.1529339. [DOI] [PubMed] [Google Scholar]
- Fisher R. P., Jin P., Chamberlin H. M., Morgan D. O. Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase. Cell. 1995 Oct 6;83(1):47–57. doi: 10.1016/0092-8674(95)90233-3. [DOI] [PubMed] [Google Scholar]
- Fisher R. P., Morgan D. O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase. Cell. 1994 Aug 26;78(4):713–724. doi: 10.1016/0092-8674(94)90535-5. [DOI] [PubMed] [Google Scholar]
- Goodrich J. A., Tjian R. Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. Cell. 1994 Apr 8;77(1):145–156. doi: 10.1016/0092-8674(94)90242-9. [DOI] [PubMed] [Google Scholar]
- Graves B. J., Johnson P. F., McKnight S. L. Homologous recognition of a promoter domain common to the MSV LTR and the HSV tk gene. Cell. 1986 Feb 28;44(4):565–576. doi: 10.1016/0092-8674(86)90266-7. [DOI] [PubMed] [Google Scholar]
- Gurdon J. B., Wickens M. P. The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 1983;101:370–386. doi: 10.1016/0076-6879(83)01028-9. [DOI] [PubMed] [Google Scholar]
- Guzder S. N., Qiu H., Sommers C. H., Sung P., Prakash L., Prakash S. DNA repair gene RAD3 of S. cerevisiae is essential for transcription by RNA polymerase II. Nature. 1994 Jan 6;367(6458):91–94. doi: 10.1038/367091a0. [DOI] [PubMed] [Google Scholar]
- Guzder S. N., Sung P., Bailly V., Prakash L., Prakash S. RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature. 1994 Jun 16;369(6481):578–581. doi: 10.1038/369578a0. [DOI] [PubMed] [Google Scholar]
- Ha I., Lane W. S., Reinberg D. Cloning of a human gene encoding the general transcription initiation factor IIB. Nature. 1991 Aug 22;352(6337):689–695. doi: 10.1038/352689a0. [DOI] [PubMed] [Google Scholar]
- Harland R. M., Weintraub H., McKnight S. L. Transcription of DNA injected into Xenopus oocytes is influenced by template topology. Nature. 1983 Mar 3;302(5903):38–43. doi: 10.1038/302038a0. [DOI] [PubMed] [Google Scholar]
- Holstege F. C., Tantin D., Carey M., van der Vliet P. C., Timmers H. T. The requirement for the basal transcription factor IIE is determined by the helical stability of promoter DNA. EMBO J. 1995 Feb 15;14(4):810–819. doi: 10.1002/j.1460-2075.1995.tb07059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humbert S., van Vuuren H., Lutz Y., Hoeijmakers J. H., Egly J. M., Moncollin V. p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair. EMBO J. 1994 May 15;13(10):2393–2398. doi: 10.1002/j.1460-2075.1994.tb06523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Izaurralde E., Lewis J., Gamberi C., Jarmolowski A., McGuigan C., Mattaj I. W. A cap-binding protein complex mediating U snRNA export. Nature. 1995 Aug 24;376(6542):709–712. doi: 10.1038/376709a0. [DOI] [PubMed] [Google Scholar]
- Izban M. G., Luse D. S. The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'----5' direction in the presence of elongation factor SII. Genes Dev. 1992 Jul;6(7):1342–1356. doi: 10.1101/gad.6.7.1342. [DOI] [PubMed] [Google Scholar]
- Jiang Y., Gralla J. D. Nucleotide requirements for activated RNA polymerase II open complex formation in vitro. J Biol Chem. 1995 Jan 20;270(3):1277–1281. doi: 10.1074/jbc.270.3.1277. [DOI] [PubMed] [Google Scholar]
- Kenny M. K., Schlegel U., Furneaux H., Hurwitz J. The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem. 1990 May 5;265(13):7693–7700. [PubMed] [Google Scholar]
- Krumm A., Meulia T., Brunvand M., Groudine M. The block to transcriptional elongation within the human c-myc gene is determined in the promoter-proximal region. Genes Dev. 1992 Nov;6(11):2201–2213. doi: 10.1101/gad.6.11.2201. [DOI] [PubMed] [Google Scholar]
- London L., Keene R. G., Landick R. Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract. Mol Cell Biol. 1991 Sep;11(9):4599–4615. doi: 10.1128/mcb.11.9.4599. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKnight S. L., Kingsbury R. Transcriptional control signals of a eukaryotic protein-coding gene. Science. 1982 Jul 23;217(4557):316–324. doi: 10.1126/science.6283634. [DOI] [PubMed] [Google Scholar]
- Meulia T., Krumm A., Groudine M. Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis. Mol Cell Biol. 1993 Sep;13(9):5647–5658. doi: 10.1128/mcb.13.9.5647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meulia T., Krumm A., Spencer C., Groudine M. Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter. Mol Cell Biol. 1992 Oct;12(10):4590–4600. doi: 10.1128/mcb.12.10.4590. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller H., Asselin C., Dufort D., Yang J. Q., Gupta K., Marcu K. B., Nepveu A. A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block. Mol Cell Biol. 1989 Dec;9(12):5340–5349. doi: 10.1128/mcb.9.12.5340. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- O'Brien T., Hardin S., Greenleaf A., Lis J. T. Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation. Nature. 1994 Jul 7;370(6484):75–77. doi: 10.1038/370075a0. [DOI] [PubMed] [Google Scholar]
- Pan G., Greenblatt J. Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site. J Biol Chem. 1994 Dec 2;269(48):30101–30104. [PubMed] [Google Scholar]
- Parvin J. D., Sharp P. A. DNA topology and a minimal set of basal factors for transcription by RNA polymerase II. Cell. 1993 May 7;73(3):533–540. doi: 10.1016/0092-8674(93)90140-l. [DOI] [PubMed] [Google Scholar]
- Parvin J. D., Timmers H. T., Sharp P. A. Promoter specificity of basal transcription factors. Cell. 1992 Mar 20;68(6):1135–1144. doi: 10.1016/0092-8674(92)90084-p. [DOI] [PubMed] [Google Scholar]
- Poon R. Y., Yamashita K., Howell M., Ershler M. A., Belyavsky A., Hunt T. Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15. J Cell Sci. 1994 Oct;107(Pt 10):2789–2799. doi: 10.1242/jcs.107.10.2789. [DOI] [PubMed] [Google Scholar]
- Price D. H., Sluder A. E., Greenleaf A. L. Dynamic interaction between a Drosophila transcription factor and RNA polymerase II. Mol Cell Biol. 1989 Apr;9(4):1465–1475. doi: 10.1128/mcb.9.4.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qiu H., Park E., Prakash L., Prakash S. The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev. 1993 Nov;7(11):2161–2171. doi: 10.1101/gad.7.11.2161. [DOI] [PubMed] [Google Scholar]
- Roberts S., Bentley D. L. Distinct modes of transcription read through or terminate at the c-myc attenuator. EMBO J. 1992 Mar;11(3):1085–1093. doi: 10.1002/j.1460-2075.1992.tb05147.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- Seroz T., Hwang J. R., Moncollin V., Egly J. M. TFIIH: a link between transcription, DNA repair and cell cycle regulation. Curr Opin Genet Dev. 1995 Apr;5(2):217–221. doi: 10.1016/0959-437x(95)80011-5. [DOI] [PubMed] [Google Scholar]
- 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]
- Strobl L. J., Eick D. Hold back of RNA polymerase II at the transcription start site mediates down-regulation of c-myc in vivo. EMBO J. 1992 Sep;11(9):3307–3314. doi: 10.1002/j.1460-2075.1992.tb05409.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tamm I. Definition of subclasses of nucleoplasmic RNA. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5011–5015. doi: 10.1073/pnas.74.11.5011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson N. E., Aronson D. B., Burgess R. R. Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody. J Biol Chem. 1990 Apr 25;265(12):7069–7077. [PubMed] [Google Scholar]
- Timmers H. T. Transcription initiation by RNA polymerase II does not require hydrolysis of the beta-gamma phosphoanhydride bond of ATP. EMBO J. 1994 Jan 15;13(2):391–399. doi: 10.1002/j.1460-2075.1994.tb06273.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Toohey M. G., Jones K. A. In vitro formation of short RNA polymerase II transcripts that terminate within the HIV-1 and HIV-2 promoter-proximal downstream regions. Genes Dev. 1989 Mar;3(3):265–282. doi: 10.1101/gad.3.3.265. [DOI] [PubMed] [Google Scholar]
- Valay J. G., Simon M., Dubois M. F., Bensaude O., Facca C., Faye G. The KIN28 gene is required both for RNA polymerase II mediated transcription and phosphorylation of the Rpb1p CTD. J Mol Biol. 1995 Jun 9;249(3):535–544. doi: 10.1006/jmbi.1995.0316. [DOI] [PubMed] [Google Scholar]
- Wang Z., Svejstrup J. Q., Feaver W. J., Wu X., Kornberg R. D., Friedberg E. C. Transcription factor b (TFIIH) is required during nucleotide-excision repair in yeast. Nature. 1994 Mar 3;368(6466):74–76. doi: 10.1038/368074a0. [DOI] [PubMed] [Google Scholar]
- Xiao H., Pearson A., Coulombe B., Truant R., Zhang S., Regier J. L., Triezenberg S. J., Reinberg D., Flores O., Ingles C. J. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol. 1994 Oct;14(10):7013–7024. doi: 10.1128/mcb.14.10.7013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yankulov K., Blau J., Purton T., Roberts S., Bentley D. L. Transcriptional elongation by RNA polymerase II is stimulated by transactivators. Cell. 1994 Jun 3;77(5):749–759. doi: 10.1016/0092-8674(94)90058-2. [DOI] [PubMed] [Google Scholar]
- Yankulov K., Yamashita K., Roy R., Egly J. M., Bentley D. L. The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase. J Biol Chem. 1995 Oct 13;270(41):23922–23925. doi: 10.1074/jbc.270.41.23922. [DOI] [PubMed] [Google Scholar]
- Zawel L., Kumar K. P., Reinberg D. Recycling of the general transcription factors during RNA polymerase II transcription. Genes Dev. 1995 Jun 15;9(12):1479–1490. doi: 10.1101/gad.9.12.1479. [DOI] [PubMed] [Google Scholar]