The transcriptional cycle of HIV-1 in real-time and live cells
RNA polymerase II (RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus (HIV) type 1 reporters, we study real-time messenger RNA (mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Thr...
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2007
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219525_v179_n2_p291_Boireau http://hdl.handle.net/20.500.12110/paper_00219525_v179_n2_p291_Boireau |
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paper:paper_00219525_v179_n2_p291_Boireau2023-06-08T14:43:40Z The transcriptional cycle of HIV-1 in real-time and live cells camptothecin messenger RNA RNA polymerase II transactivator protein virus RNA article biogenesis chromatin immunoprecipitation enzyme activity fluorescence recovery after photobleaching human human cell Human immunodeficiency virus 1 Human immunodeficiency virus 1 infection in situ hybridization kinetics life cycle nonhuman nucleotide sequence priority journal promoter region transcription initiation virus cell interaction virus transcription Cell Line, Tumor Cell Survival Computer Simulation Fluorescence Recovery After Photobleaching Gene Expression Regulation, Viral Genes, Reporter HIV-1 Humans In Situ Hybridization Kinetics Models, Genetic Mutation Photobleaching Polyadenylation RNA 3' End Processing RNA Polymerase II RNA, Messenger RNA, Viral Time Factors Transcription, Genetic Human immunodeficiency virus 1 RNA polymerase II (RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus (HIV) type 1 reporters, we study real-time messenger RNA (mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Through modeling, the use of mutant polymerases, drugs, and quantitative in situ hybridiza tion, we investigate the kinetics of the HIV-1 transcription cycle. Initiation appears efficient because most polymerases demonstrate stable gene association. We calculate an elongation rate of approximately 1.9 kb/min, and, surprisingly, polymerases remain at transcription sites 2.5 min longer than nascent RNAs. With a total polymerase residency time estimated at 333 s, 114 are assigned to elongation, and 63 are assigned to 3′-end processing and/or transcript release. However, mRNAs were released seconds after polyadenylation onset, and analysis of polymerase density by chromatin immunoprecipitation suggests that they pause or lose processivity after passing the polyA site. The strengths and limitations of this kinetic approach to analyze mRNA biogenesis in living cells are discussed. © The Rockefeller University Press. 2007 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219525_v179_n2_p291_Boireau http://hdl.handle.net/20.500.12110/paper_00219525_v179_n2_p291_Boireau |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
camptothecin messenger RNA RNA polymerase II transactivator protein virus RNA article biogenesis chromatin immunoprecipitation enzyme activity fluorescence recovery after photobleaching human human cell Human immunodeficiency virus 1 Human immunodeficiency virus 1 infection in situ hybridization kinetics life cycle nonhuman nucleotide sequence priority journal promoter region transcription initiation virus cell interaction virus transcription Cell Line, Tumor Cell Survival Computer Simulation Fluorescence Recovery After Photobleaching Gene Expression Regulation, Viral Genes, Reporter HIV-1 Humans In Situ Hybridization Kinetics Models, Genetic Mutation Photobleaching Polyadenylation RNA 3' End Processing RNA Polymerase II RNA, Messenger RNA, Viral Time Factors Transcription, Genetic Human immunodeficiency virus 1 |
| spellingShingle |
camptothecin messenger RNA RNA polymerase II transactivator protein virus RNA article biogenesis chromatin immunoprecipitation enzyme activity fluorescence recovery after photobleaching human human cell Human immunodeficiency virus 1 Human immunodeficiency virus 1 infection in situ hybridization kinetics life cycle nonhuman nucleotide sequence priority journal promoter region transcription initiation virus cell interaction virus transcription Cell Line, Tumor Cell Survival Computer Simulation Fluorescence Recovery After Photobleaching Gene Expression Regulation, Viral Genes, Reporter HIV-1 Humans In Situ Hybridization Kinetics Models, Genetic Mutation Photobleaching Polyadenylation RNA 3' End Processing RNA Polymerase II RNA, Messenger RNA, Viral Time Factors Transcription, Genetic Human immunodeficiency virus 1 The transcriptional cycle of HIV-1 in real-time and live cells |
| topic_facet |
camptothecin messenger RNA RNA polymerase II transactivator protein virus RNA article biogenesis chromatin immunoprecipitation enzyme activity fluorescence recovery after photobleaching human human cell Human immunodeficiency virus 1 Human immunodeficiency virus 1 infection in situ hybridization kinetics life cycle nonhuman nucleotide sequence priority journal promoter region transcription initiation virus cell interaction virus transcription Cell Line, Tumor Cell Survival Computer Simulation Fluorescence Recovery After Photobleaching Gene Expression Regulation, Viral Genes, Reporter HIV-1 Humans In Situ Hybridization Kinetics Models, Genetic Mutation Photobleaching Polyadenylation RNA 3' End Processing RNA Polymerase II RNA, Messenger RNA, Viral Time Factors Transcription, Genetic Human immunodeficiency virus 1 |
| description |
RNA polymerase II (RNAPII) is a fundamental enzyme, but few studies have analyzed its activity in living cells. Using human immunodeficiency virus (HIV) type 1 reporters, we study real-time messenger RNA (mRNA) biogenesis by photobleaching nascent RNAs and RNAPII at specific transcription sites. Through modeling, the use of mutant polymerases, drugs, and quantitative in situ hybridiza tion, we investigate the kinetics of the HIV-1 transcription cycle. Initiation appears efficient because most polymerases demonstrate stable gene association. We calculate an elongation rate of approximately 1.9 kb/min, and, surprisingly, polymerases remain at transcription sites 2.5 min longer than nascent RNAs. With a total polymerase residency time estimated at 333 s, 114 are assigned to elongation, and 63 are assigned to 3′-end processing and/or transcript release. However, mRNAs were released seconds after polyadenylation onset, and analysis of polymerase density by chromatin immunoprecipitation suggests that they pause or lose processivity after passing the polyA site. The strengths and limitations of this kinetic approach to analyze mRNA biogenesis in living cells are discussed. © The Rockefeller University Press. |
| title |
The transcriptional cycle of HIV-1 in real-time and live cells |
| title_short |
The transcriptional cycle of HIV-1 in real-time and live cells |
| title_full |
The transcriptional cycle of HIV-1 in real-time and live cells |
| title_fullStr |
The transcriptional cycle of HIV-1 in real-time and live cells |
| title_full_unstemmed |
The transcriptional cycle of HIV-1 in real-time and live cells |
| title_sort |
transcriptional cycle of hiv-1 in real-time and live cells |
| publishDate |
2007 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00219525_v179_n2_p291_Boireau http://hdl.handle.net/20.500.12110/paper_00219525_v179_n2_p291_Boireau |
| _version_ |
1768546660661592064 |