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- Title
The transcriptional cycle of HIV-1 in real-time and live cells.
- Authors
Boireau, Stéphanie; Maiuri, Paolo; Basyuk, Eugenia; de la Mata, Manuel; Knezevich, Anna; Pradet-Balade, Bérangère; Bäcker, Volker; Kornblihtt, Alberto; Marcello, Alessandro; Bertrand, Edouard
- 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.
- Subjects
GENETIC transcription; HIV; MESSENGER RNA; REPORTER genes; RNA polymerases; CHROMATIN
- Publication
Journal of Cell Biology, 2007, Vol 179, Issue 2, p291
- ISSN
0021-9525
- Publication type
Article
- DOI
10.1083/jcb.200706018