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The transcription cycle of RNA polymerase II in living cells
RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polym...
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2002
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173384/ https://www.ncbi.nlm.nih.gov/pubmed/12473686 http://dx.doi.org/10.1083/jcb.200206019 |
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author | Kimura, Hiroshi Sugaya, Kimihiko Cook, Peter R. |
author_facet | Kimura, Hiroshi Sugaya, Kimihiko Cook, Peter R. |
author_sort | Kimura, Hiroshi |
collection | PubMed |
description | RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: ∼75% moved rapidly, but ∼25% was transiently immobile (association t(1/2) ≈ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [(3)H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t(1/2) ≈ 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases. |
format | Text |
id | pubmed-2173384 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2002 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-21733842008-05-01 The transcription cycle of RNA polymerase II in living cells Kimura, Hiroshi Sugaya, Kimihiko Cook, Peter R. J Cell Biol Article RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: ∼75% moved rapidly, but ∼25% was transiently immobile (association t(1/2) ≈ 20 min) and transcriptionally active, as incubation with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole eliminated it. No immobile but inactive fraction was detected, providing little support for the existence of a stable holoenzyme, or the slow stepwise assembly of a preinitiation complex on promoters or the nuclear substructure. Actinomycin D decreased the rapidly moving fraction, suggesting that engaged polymerases stall at intercalated molecules while others initiate. When wild-type cells containing only the endogenous enzyme were incubated with [(3)H]uridine, nascent transcripts became saturated with tritium with similar kinetics (t(1/2) ≈ 14 min). These data are consistent with a polymerase being mobile for one half to five sixths of a transcription cycle, and rapid assembly into the preinitiation complex. Then, most expressed transcription units would spend significant times unassociated with engaged polymerases. The Rockefeller University Press 2002-12-09 /pmc/articles/PMC2173384/ /pubmed/12473686 http://dx.doi.org/10.1083/jcb.200206019 Text en Copyright © 2002, The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Article Kimura, Hiroshi Sugaya, Kimihiko Cook, Peter R. The transcription cycle of RNA polymerase II in living cells |
title | The transcription cycle of RNA polymerase II in living cells |
title_full | The transcription cycle of RNA polymerase II in living cells |
title_fullStr | The transcription cycle of RNA polymerase II in living cells |
title_full_unstemmed | The transcription cycle of RNA polymerase II in living cells |
title_short | The transcription cycle of RNA polymerase II in living cells |
title_sort | transcription cycle of rna polymerase ii in living cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173384/ https://www.ncbi.nlm.nih.gov/pubmed/12473686 http://dx.doi.org/10.1083/jcb.200206019 |
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