Transcription Dynamics at the HIV-1 Reporter Locus

Abstract

Transcription is a centerpiece in the regulation of gene expression and cell metabolism. It can be divided in three stages: initiation, elongation and termination. The molecular mechanisms and steps involved in the regulation of transcription are of a dynamic nature. Decades ago, biochemistry techniques could not tell us much about the kinetics of transcription. However, recent advances in microscopy make it feasible to visualize and quantify transcription in live cells at the single-cell and single-molecule levels. With state-of-the art live cell microscopy, it is now possible to observe fluctuations in transcription kinetics at single copy genes. Using an established cell line and our Fabs (Fragment Antigen-Binding) for live imaging of RNAPII, we have imaged transcription dynamics at the locus of a reporter gene controlled by the HIV-1 promoter and tagged with 128x MS2 cassette, when transcribed in RNA, the MS2 repeats are bound by MCP-GFP. The MS2 bright spot at the reporter locus provides information about the gene location and transcription readout, while our Fabs, C13 and Pa57 tell us about the recruitment of the RNAPII to the promoter and initiation of transcription, respectively. Here, simultaneous imaging of transcription (MS2), RNAPII recruitment (C13) and initiation (Pa57) dynamics show temporal correlations at the reporter locus.