Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

To investigate functional differences between RNA polymerases IIA and IIO (Pol IIA and Pol IIO), with hypo- and hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes. Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol IIO arrayed in markedly different, locus- and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol IIO, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol IIO, suggesting that heat shock genes are transcribed by a mixture of hypo- and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol IIO.

[1]  David M. Chao,et al.  A multisubunit complex associated with the RNA polymerase II CTD and TATA-binding protein in yeast , 1993, Cell.

[2]  R. Conaway,et al.  Phosphorylation of C-terminal domain of RNA polymerase II is not required in basal transcription , 1993, Nature.

[3]  A. Dvir,et al.  Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. , 1993, The Journal of biological chemistry.

[4]  D. Spector,et al.  In vivo evidence that transcription and splicing are coordinated by a recruiting mechanism , 1993, Cell.

[5]  A. Greenleaf,et al.  Positive patches and negative noodles: linking RNA processing to transcription? , 1993, Trends in biochemical sciences.

[6]  J. Lawrence,et al.  Higher level organization of individual gene transcription and RNA splicing. , 1993, Science.

[7]  J. Wiśniewski,et al.  Regulation of heat shock factor trimer formation: role of a conserved leucine zipper. , 1993, Science.

[8]  M. Dahmus,et al.  Partial purification and characterization of two distinct protein kinases that differentially phosphorylate the carboxyl-terminal domain of RNA polymerase subunit IIa. , 1993, The Journal of biological chemistry.

[9]  J. Lis,et al.  Promoter melting and TFIID complexes on Drosophila genes in vivo. , 1992, Genes & development.

[10]  R. Kornberg,et al.  Cloning of a subunit of yeast RNA polymerase II transcription factor b and CTD kinase. , 1992, Science.

[11]  D. Reinberg,et al.  Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II , 1992, Nature.

[12]  R. Conaway,et al.  A carboxyl-terminal-domain kinase associated with RNA polymerase II transcription factor delta from rat liver. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[13]  D. Reinberg,et al.  Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein , 1992, Cell.

[14]  R. Young,et al.  A novel transcription factor reveals a functional link between the RNA polymerase II CTD and TFIID , 1992, Cell.

[15]  J. Chesnut,et al.  The interaction of RNA polymerase II with the adenovirus-2 major late promoter is precluded by phosphorylation of the C-terminal domain of subunit IIa. , 1992, The Journal of biological chemistry.

[16]  P. Farnham,et al.  The HIP1 initiator element plays a role in determining the in vitro requirement of the dihydrofolate reductase gene promoter for the C-terminal domain of RNA polymerase II , 1992, Molecular and cellular biology.

[17]  J. Ausió,et al.  Chromatin dynamics and the modulation of genetic activity. , 1992, Trends in biochemical sciences.

[18]  D. Reinberg,et al.  Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II. , 1992, The Journal of biological chemistry.

[19]  J. Lis,et al.  DNA sequence requirements for generating paused polymerase at the start of hsp70. , 1992, Genes & development.

[20]  G. Dreyfuss,et al.  Isolation of hnRNP complexes from Drosophila melanogaster , 1992, The Journal of cell biology.

[21]  D. Reinberg,et al.  The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Carl Wu,et al.  Stress-induced oligomerization and chromosomal relocalization of heat-shock factor , 1991, Nature.

[23]  J. Lis,et al.  RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene , 1991, Molecular and cellular biology.

[24]  D. Withers,et al.  The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. , 1991, Genetics.

[25]  J. Gall,et al.  Spliceosomes and snurposomes , 1991, Science.

[26]  J. M. Lee,et al.  CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae. , 1991, Gene expression.

[27]  I. Herskowitz,et al.  A functional interaction between the C-terminal domain of RNA polymerase II and the negative regulator SIN1 , 1991, Cell.

[28]  J Zhang,et al.  Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. , 1991, The Journal of biological chemistry.

[29]  R. Paro,et al.  Direct interaction of the Polycomb protein with Antennapedia regulatory sequences in polytene chromosomes of Drosophila melanogaster. , 1991, The EMBO journal.

[30]  C. Thummel,et al.  Splicing precedes polyadenylation during Drosophila E74A transcription , 1990, Molecular and cellular biology.

[31]  A. Rougvie,et al.  Postinitiation transcriptional control in Drosophila melanogaster , 1990, Molecular and cellular biology.

[32]  R. Young,et al.  RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals , 1990, Nature.

[33]  M. Morange,et al.  Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. , 1990, European journal of biochemistry.

[34]  J. Corden.  Tails of RNA polymerase II. , 1990, Trends in biochemical sciences.

[35]  P. Sharp,et al.  Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit , 1990, Molecular and cellular biology.

[36]  M. Morange,et al.  Heat-shock and related stress enhance RNA polymerase II C-terminal domain kinase activity in HeLa cell extracts , 1990 .

[37]  P. Laybourn,et al.  Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription. , 1990, The Journal of biological chemistry.

[38]  A. Greenleaf,et al.  The carboxyl-terminal repeat domain of RNA polymerase II is not required for transcription factor Sp1 to function in vitro. , 1990, The Journal of biological chemistry.

[39]  R. Young,et al.  RNA polymerase II subunit composition, stoichiometry, and phosphorylation , 1990, Molecular and cellular biology.

[40]  D. Hogness,et al.  The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins , 1990, Cell.

[41]  D. Hogness,et al.  Spatial and temporal patterns of E74 transcription during Drosophila development , 1990, Cell.

[42]  D. Hogness,et al.  The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. , 1990, Genes & development.

[43]  T. Guilfoyle.  A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II. , 1989, The Plant cell.

[44]  N. Thompson,et al.  Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. , 1989, The Journal of biological chemistry.

[45]  J. M. Lee,et al.  A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[46]  A. Stevens,et al.  5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole inhibits a HeLa protein kinase that phosphorylates an RNA polymerase II-derived peptide☆ , 1989 .

[47]  A. Rougvie,et al.  The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged , 1988, Cell.

[48]  David A. Agard,et al.  Precise determination of the molecular limits of a polytene chromosome band: Regulatory sequences for the Notch gene are in the interband , 1988, Cell.

[49]  Y. Osheim,et al.  Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. , 1988, Genes & development.

[50]  J. M. Lee,et al.  The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[51]  H. Xiao,et al.  Germline transformation used to define key features of heat-shock response elements. , 1988, Science.

[52]  C. Ingles,et al.  The C-terminal domain of the largest subunit of RNA polymerase II of Saccharomyces cerevisiae, Drosophila melanogaster, and mammals: a conserved structure with an essential function , 1988, Molecular and cellular biology.

[53]  M. Bartolomei,et al.  Genetic analysis of the repetitive carboxyl-terminal domain of the largest subunit of mouse RNA polymerase II , 1988, Molecular and cellular biology.

[54]  M. Dahmus,et al.  Messenger RNA synthesis in mammalian cells is catalyzed by the phosphorylated form of RNA polymerase II. , 1987, The Journal of biological chemistry.

[55]  R. Young,et al.  Functional redundancy and structural polymorphism in the large subunit of RNA polymerase II , 1987, Cell.

[56]  J. Lis,et al.  RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. , 1986, Molecular and cellular biology.

[57]  C. Meares,et al.  RNA contacts subunits IIo and IIc in HeLa RNA polymerase II transcription complexes. , 1986, The Journal of biological chemistry.

[58]  W. Kim,et al.  Immunochemical analysis of mammalian RNA polymerase II subspecies. Stability and relative in vivo concentration. , 1986, The Journal of biological chemistry.

[59]  Carl Wu.  Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin , 1984, Nature.

[60]  J. Lis,et al.  New heat shock puffs and β=galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybrid gene , 1983, Cell.

[61]  A. Greenleaf,et al.  Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme. , 1982, The Journal of biological chemistry.

[62]  F. Jensen,et al.  Anti-RNP monoclonal antibodies derived from a mouse strain with lupus-like autoimmunity. , 1982, Journal of immunology.

[63]  A. Greenleaf,et al.  α-amanitin-resistant D. melanogaster with an altered RNA polymerase II , 1979, Cell.

[64]  M. Ashburner,et al.  The induction of gene activity in drosophila by heat shock , 1979, Cell.

[65]  A. Greenleaf,et al.  Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[66]  L. Silver,et al.  A method for determination of the in situ distribution of chromosomal proteins. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[67]  P. Agris,et al.  Immunochemical analysis of an arginine-rich systemic lupus erythematosus autoepitope. , 1993, Autoimmunity.

[68]  W. Dynan,et al.  5 Phosphorylation of RNA Polymerase II as a Transcriptional Regulatory Mechanism , 1992 .

[69]  S. Elgin,et al.  Distribution of chromosomal proteins in polytene chromosomes of Drosophila. , 1991, Methods in cell biology.

[70]  R. Young,et al.  RNA polymerase II. , 1991, Annual review of biochemistry.

[71]  R. Tjian,et al.  Affinity-purified antibody as a probe of RNA polymerase II subunit structure. , 1984, Journal of molecular and applied genetics.

[72]  A. Greenleaf,et al.  Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II. , 1979, Cell.

[73]  A. Greenleaf,et al.  LOCALIZATION OF RNA POLYMERASE ON DROSOPHILA POLYTENE CHROMOSOMES BY INDIRECT IMMUNOFLUORESCENCE , 1976 .

[74]  J. Simon,et al.  Localization and expression of transformed DNA sequences within heat shock puffs of Drosophila melanogaster , 2022 .