Institut de Génétique Moléculaire de Montpellier, CNRS UMR5535, 1919, route de Mende, 34293 Montpellier Cedex 5, France.
Unité Imagerie et Modélisation, Département Biologie Cellulaire et Infections, Institut Pasteur and CNRS UMR 3691, 28, rue du Docteur Roux, 75015 Paris, France.
Nat Commun. 2016 Jul 27;7:12248. doi: 10.1038/ncomms12248.
Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.
活细胞成像揭示了基因表达的意外特征。在这里，我们使用改进的单分子 RNA 显微镜技术，表明 HIV-1 RNA 的合成是由一群紧密间隔的聚合酶（称为车队）实现的，而不是单个孤立的酶。车队是通过中介体依赖性重新起始机制产生的，该机制产生了一个短暂但快速的聚合酶起始和逃离启动子的连续过程。在延伸过程中，聚合酶之间的间隔只有几百个核苷酸，物理建模表明 DNA 扭结应力可能维持聚合酶的间隔。我们还观察到 HIV-1 启动子在两个时间尺度上表现出随机波动，我们称之为多尺度爆发。每个时间尺度都是独立调节的：中介体控制分钟级波动（车队），而 TBP-TATA 盒相互作用控制亚小时波动（长许可/非许可期）。细胞启动子也产生聚合酶车队，并显示多尺度爆发。我们提出，缓慢的、依赖 TBP 的波动对单细胞的表型变异性很重要。
