Mitotic repression of transcription in vitro.
A normal consequence of mitosis in eukaryotes is the repression of transcription. Using Xenopus egg extracts shifted to a mitotic state by the addition of purified cyclin, we have for the first time been able to reproduce a mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts, but strongly repressed in extracts converted to mitosis. With the topoisomerase II inhibitor VM-26, we demonstrate that this mitotic repression of RNA polymerase III transcription does not require normal chromatin condensation. Similarly; in vitro mitotic repression of transcription does not require the presence of nucleosome structure or involve a general repressive chromatin-binding protein, as inhibition of chromatin formation with saturating amounts of non-specific DNA has no effect on repression. Instead, the mitotic repression of transcription appears to be due to phosphorylation of a component of the transcription machinery by a mitotic protein kinase, either cdc2 kinase and/or a kinase activated by it. Mitotic repression of RNA polymerase III transcription is observed both in complete mitotic cytosol and when a kinase-enriched mitotic fraction is added to a highly simplified 5S RNA transcription reaction. We present evidence that, upon depletion of cdc2 kinase, a secondary protein kinase activity remains and can mediate this in vitro mitotic repression of transcription.
Hartl
P
P
Department of Biology, University of California, San Diego, La Jolla 92093-0322.
Gottesfeld
J
J
Forbes
D J
DJ
D000818:Animals; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism; D002474:Cell-Free System; D016213:Cyclins / Q000494:pharmacology; D005260:Female; D007399:Interphase / Q000502:physiology; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D008954:Models, Biological; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism; D012320:RNA Polymerase III / Q000378:metabolism; D013698:Templates, Genetic; D013713:Teniposide / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014982:Xenopus laevis
D016213:Cyclins; D059005:Topoisomerase II Inhibitors; D014157:Transcription Factors; D013713:Teniposide; D011494:Protein Kinases; D006656:Protamine Kinase; D016203:CDC2 Protein Kinase; D012320:RNA Polymerase III