Identification of multiple cyclin subunits of human P-TEFb.

The transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter.

[1]  J. Bazan,et al.  Helical fold prediction for the cyclin box , 1996, Proteins.

[2]  Ping Wei,et al.  A Novel CDK9-Associated C-Type Cyclin Interacts Directly with HIV-1 Tat and Mediates Its High-Affinity, Loop-Specific Binding to TAR RNA , 1998, Cell.

[3]  V. Kidd,et al.  Alternatively spliced cyclin C mRNA is widely expressed, cell cycle regulated, and encodes a truncated cyclin box. , 1996, Oncogene.

[4]  D. Price,et al.  Purification of an RNA Polymerase II Transcript Release Factor from Drosophila(*) , 1996, The Journal of Biological Chemistry.

[5]  D. Price,et al.  Control of formation of two distinct classes of RNA polymerase II elongation complexes , 1992, Molecular and cellular biology.

[6]  R. Conaway,et al.  The RNA polymerase II general elongation factors. , 1996, Trends in biochemical sciences.

[7]  F. Winston,et al.  Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in Saccharomyces cerevisiae. , 1998, Genes & development.

[8]  D. Price,et al.  Purification of P-TEFb, a Transcription Factor Required for the Transition into Productive Elongation (*) , 1995, The Journal of Biological Chemistry.

[9]  M. Dahmus.  Phosphorylation of the C-terminal domain of RNA polymerase II. , 1995, Biochimica et biophysica acta.

[10]  D. Hazuda,et al.  P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. , 1997, Genes & development.

[11]  K. Yano,et al.  DSIF, a novel transcription elongation factor that regulates RNA polymerase II processivity, is composed of human Spt4 and Spt5 homologs. , 1998, Genes & development.

[12]  T. Hodge,et al.  Host genes and HIV: the role of the chemokine receptor gene CCR5 and its allele. , 1997, Emerging infectious diseases.

[13]  R. Müller,et al.  Cyclin ET, a new splice variant of human cyclin E with a unique expression pattern during cell cycle progression and differentiation. , 1997, Nucleic acids research.

[14]  D O Morgan,et al.  Cyclin-dependent kinases: engines, clocks, and microprocessors. , 1997, Annual review of cell and developmental biology.

[15]  David O. Morgan,et al.  Principles of CDK regulation , 1995, Nature.

[16]  M. Mathews,et al.  Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro. , 1997, Genes & development.

[17]  H. Handa,et al.  Interplay between positive and negative elongation factors: drawing a new view of DRB , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[18]  R. Müller,et al.  Alternative splicing of human cyclin E. , 1994, Journal of cell science.

[19]  X. Graña,et al.  The CDC2-related kinase PITALRE is the catalytic subunit of active multimeric protein complexes. , 1996, The Biochemical journal.

[20]  D. Price,et al.  Control of RNA Polymerase II Elongation Potential by a Novel Carboxyl-terminal Domain Kinase* , 1996, The Journal of Biological Chemistry.

[21]  B. Peterlin,et al.  Effects of human chromosome 12 on interactions between Tat and TAR of human immunodeficiency virus type 1 , 1994, Journal of virology.

[22]  P. Laybourn,et al.  Transcription-dependent structural changes in the C-terminal domain of mammalian RNA polymerase subunit IIa/o. , 1989, The Journal of biological chemistry.

[23]  D. Littman,et al.  Expression cloning of new receptors used by simian and human immunodeficiency viruses , 1997, Nature.

[24]  M. Dahmus.  The role of multisite phosphorylation in the regulation of RNA polymerase II activity. , 1994, Progress in nucleic acid research and molecular biology.

[25]  B. Peterlin,et al.  Human chromosome 12 is required for optimal interactions between Tat and TAR of human immunodeficiency virus type 1 in rodent cells , 1992, Journal of virology.

[26]  D. Price,et al.  Unusual Nucleic Acid Binding Properties of Factor 2, an RNA Polymerase II Transcript Release Factor* , 1998, The Journal of Biological Chemistry.

[27]  D. Price,et al.  Drosophila Factor 2, an RNA Polymerase II Transcript Release Factor, Has DNA-dependent ATPase Activity* , 1997, The Journal of Biological Chemistry.

[28]  J. Lis,et al.  Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation , 1994, Nature.

[29]  A. Giordano,et al.  PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro. , 1994, Proceedings of the National Academy of Sciences of the United States of America.