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¹ Institute of Molecular Genetics of Montpellier, Unité Mixte de Recherche 5535, and ² Montpellier Rio Imaging, Centre National de la Recherche Scientifique, 34293 Montpellier, France
³ Laboratory of Molecular Virology, International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy
⁴ Laboratorio de Fisiología y Biología Molecular, Departamento de Fisiología, Biología Molecular, y Celular, Instituto de Fisiología, Biología Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, (C1428EHA) Buenos Aires, Argentina

Correspondence to Alessandro Marcello: Marcello{at}icgeb.org; or Edouard Bertrand: Edouard.Bertrand{at}igmm.cnrs.fr

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.

S. Boireau, P. Maiuri, and E. Basyuk contributed equally to this paper.

Abbreviations used in this paper: CTD, C-terminal domain; HIV, human immunodeficiency virus; LTR, long terminal repeat; RNAPII, RNA polymerase II; WT, wild type.

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