Complexity of RNA polymerase II elongation dynamics

Abstract

Transcription of protein-coding genes by RNA polymerase II can be regulated at multiple points during the process of RNA synthesis, including initiation, elongation, and termination. In vivo data suggests that elongating polymerases exhibit heterogeneity throughout the gene body, suggestive of changes in elongation rate and/or pausing. Here, we review evidence from a variety of different experimental approaches for understanding regulation of transcription elongation. We compare steady-state measurements of nascent RNA density and polymerase occupancy to time-resolved measurements and point out areas of disagreement. Finally, we discuss future avenues of investigation for understanding this critically important step in gene regulation. This article is part of a Special Issue entitled: Chromatin in time and space.

Fig. 1.

Live cell kinetic measurements of transcription elongation. A) Pre-mRNA can be visualized using the MS2 bacteriophage capsid protein. DNA which encodes for RNA stem loops is inserted into the reporter gene of interest [51]. Once transcribed, the stem loops bind the constitutively-expressed fluorescently-labeled capsid protein. Each RNA stem loop binds a dimer of the capsid protein. The nascent RNA is then visible at the active locus in the living cell. B) Fluorescence recovery after photobleaching (FRAP) of nascent RNA. The fluorescence recovers on a time scale determined by both elongation and termination [55]. The slope of the curve depends on the length of the MS2 array, the length of the gene after the MS2 array, the velocity of the polymerase, and the termination time. The FRAP curves display the recovery dependence for different termination times (T) at a fixed velocity (v). C) Fluctuation analysis on single yeast genes [56]. Using time-lapse imaging coupled with automated tracking enables the determination of the transcription autocorrelation (red circles). The decay depends only on the dwell time of the nascent RNA (τ), and the amplitude is the product of the initiation rate (c) and the dwell time. The original data is from [55,56].

Fig. 2.

Steady-state measurements for determining the relative balance of initiation, elongation, and termination. A) Cartoon of a nascent RNA abundance measurement. The position of a PCR amplicon or FISH probe used to quantify abundance (m) is shown by the red and gray bars. In this instance, m₁=2; m₂=1. The distance from the position of the polymerase to the end of the gene is shown by l_1,2. B) Length dependence of nascent RNA abundance. As the distance to the 3′ end increases, the time that a nascent RNA is present increases (red line). The slope is the ratio of initiation rate (c) to velocity (v); the offset is the product of the initiation rate (c) and the termination time (T).