The transcriptional cycle of HIV-1 in real-time and live cells.

@article{citeulike:1902642, abstract = {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 hybridization, 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.}, address = {Institute of Molecular Genetics of Montpellier, Unit\'{e} Mixte de Recherche 5535, and 2Montpellier Rio Imaging, Centre National de la Recherche Scientifique, 34293 Montpellier, France.}, author = {Boireau, S. and Maiuri, P. and Basyuk, E. and de la Mata, M. and Knezevich, A. and Pradet-Balade, B. and B\"{a}cker, V. and Kornblihtt, A. and Marcello, A. and Bertrand, E. }, citeulike-article-id = {1902642}, doi = {http://dx.doi.org/10.1083/jcb.200706018}, issn = {0021-9525}, journal = {J Cell Biol}, keywords = {elongation, experiment, pauses, transcription}, month = {October}, number = {2}, pages = {291--304}, posted-at = {2008-07-10 17:01:41}, priority = {2}, title = {The transcriptional cycle of HIV-1 in real-time and live cells.}, url = {http://dx.doi.org/10.1083/jcb.200706018}, volume = {179}, year = {2007} }

TY - JOUR ID - citeulike:1902642 L3 - citeulike-article-id:1902642 TI - The transcriptional cycle of HIV-1 in real-time and live cells. JF - J Cell Biol VL - 179 IS - 2 SP - 291 EP - 304 AD - Institute of Molecular Genetics of Montpellier, Unité Mixte de Recherche 5535, and 2Montpellier Rio Imaging, Centre National de la Recherche Scientifique, 34293 Montpellier, France. SN - 0021-9525 N2 - 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 hybridization, 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. KW - elongation KW - experiment KW - pauses KW - transcription AU - Boireau, S AU - Maiuri, P AU - Basyuk, E AU - de la Mata, M AU - Knezevich, A AU - Pradet-Balade, B AU - Bäcker, V AU - Kornblihtt, A AU - Marcello, A AU - Bertrand, E PY - 2007/10/22/ UR - http://dx.doi.org/10.1083/jcb.200706018 ER -