The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding prote... more The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription
Research minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering t... more Research minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for Institute, 10666 North Torrey their effects on the binding affinity of full-length TFIIIA and a polypeptide Pines Road, La Jolla consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease CA 92037, U.S.A. footprinting was used to compare the binding sites of TFIIIA and zf4-7 2 Department of Biochemistry on 5 S RNA. The consistency between the data obtained from these and Microbiology, University two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA of Victoria, PO Box 3055 bind to a central core region on the 5 S RNA molecule consisting of loop Victoria, British Columbia B/helix II/loop A/helix V/region E. This information was used to design Canada V8W 3P6
). Until recently, mitotic repression was thought Nuclear transcription is repressed when eukaryo... more ). Until recently, mitotic repression was thought Nuclear transcription is repressed when eukaryotic to be primarily due to the condensation of interphase cells enter mitosis. Using Xenopus egg extracts shifted chromatin into mitotic chromosomes . However, reto the mitotic state with recombinant cyclin B1 procent studies have shown that many levels of control tein, we have been able to reproduce mitotic represare responsible for silencing transcription at mitosis, sion of transcription in vitro. Active RNA polymerase including changes in chromatin structure and III transcription is observed in interphase extracts in occupancy of promoter elements by general and genethe absence of added cyclin, but is strongly repressed specific transcription factors [7, 10]. Loss of transcripby the induction of cdc2/cyclin B (maturation/mitosis tion factors from promoter elements may be due, at promoting factor, MPF) kinase activity in the mitotic least in part, to the inhibition of the DNA binding activextract. Studies with protein kinase inhibitors show ity of gene-specific protein factors by specific phosphorthat protein phosphorylation is required for represylation events at mitosis [7,[11][12][13]. Evidence for presion. Add-back experiments indicate that repression mature termination of transcription of both messenger of class III gene transcription is due to inactivation of RNA-coding genes and ribosomal RNA genes at mitosis the transcription factor TFIIIB. TFIIIB is composed of has also been presented 15]. Studies with simplithe TATA-box binding protein (TBP) and TBP-assocified systems reveal that reversible modifications of the
Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of tra... more Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclindependent kinase inhibitor p21 Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.
The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding prote... more The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription
Research minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering t... more Research minimal polypeptide necessary for high-affinity binding to 5 S RNA. Mutations covering the entire 5 S RNA structure have been compared for Institute, 10666 North Torrey their effects on the binding affinity of full-length TFIIIA and a polypeptide Pines Road, La Jolla consisting of fingers 4 to 7 of TFIIIA (zf4-7). In addition, ribonuclease CA 92037, U.S.A. footprinting was used to compare the binding sites of TFIIIA and zf4-7 2 Department of Biochemistry on 5 S RNA. The consistency between the data obtained from these and Microbiology, University two approaches provided a clear indication that zinc fingers 4 to 7 of TFIIIA of Victoria, PO Box 3055 bind to a central core region on the 5 S RNA molecule consisting of loop Victoria, British Columbia B/helix II/loop A/helix V/region E. This information was used to design Canada V8W 3P6
). Until recently, mitotic repression was thought Nuclear transcription is repressed when eukaryo... more ). Until recently, mitotic repression was thought Nuclear transcription is repressed when eukaryotic to be primarily due to the condensation of interphase cells enter mitosis. Using Xenopus egg extracts shifted chromatin into mitotic chromosomes . However, reto the mitotic state with recombinant cyclin B1 procent studies have shown that many levels of control tein, we have been able to reproduce mitotic represare responsible for silencing transcription at mitosis, sion of transcription in vitro. Active RNA polymerase including changes in chromatin structure and III transcription is observed in interphase extracts in occupancy of promoter elements by general and genethe absence of added cyclin, but is strongly repressed specific transcription factors [7, 10]. Loss of transcripby the induction of cdc2/cyclin B (maturation/mitosis tion factors from promoter elements may be due, at promoting factor, MPF) kinase activity in the mitotic least in part, to the inhibition of the DNA binding activextract. Studies with protein kinase inhibitors show ity of gene-specific protein factors by specific phosphorthat protein phosphorylation is required for represylation events at mitosis [7,[11][12][13]. Evidence for presion. Add-back experiments indicate that repression mature termination of transcription of both messenger of class III gene transcription is due to inactivation of RNA-coding genes and ribosomal RNA genes at mitosis the transcription factor TFIIIB. TFIIIB is composed of has also been presented 15]. Studies with simplithe TATA-box binding protein (TBP) and TBP-assocified systems reveal that reversible modifications of the
Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of tra... more Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclindependent kinase inhibitor p21 Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.
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Papers by Anne Leresche