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. 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.
Boireau
Stéphanie
S
Institute of Molecular Genetics of Montpellier, Unité Mixte de Recherche 5535, Centre National de la Recherche Scientifique, 34293 Montpellier, France.
Maiuri
Paolo
P
Basyuk
Eugenia
E
de la Mata
Manuel
M
Knezevich
Anna
A
Pradet-Balade
Bérangère
B
Bäcker
Volker
V
Kornblihtt
Alberto
A
Marcello
Alessandro
A
Bertrand
Edouard
E
D045744:Cell Line, Tumor; D002470:Cell Survival; D003198:Computer Simulation; D036681:Fluorescence Recovery After Photobleaching; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D017403:In Situ Hybridization; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D038761:Photobleaching; D026723:Polyadenylation; D026721:RNA 3' End Processing; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D013997:Time Factors; D014158:Transcription, Genetic
D012333:RNA, Messenger; D012367:RNA, Viral; D012319:RNA Polymerase II