Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

Abstract

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.

Keywords: RNA polymerase II; escape to productive elongation; nascent RNA; photoactivation; promoter-proximal pausing; termination.

Figure 1.

Imaging the stability of paused Pol II at the uninduced Hsp70 transgene in living cells. (A) Schematic of the live-cell imaging experiment showing mCherry-LacI bound to the operator sites (red) and photoactivated Pol II (green). Scs and scs′ are the insulator elements flanking the Hsp70 gene. (B) Uninduced mCherry-marked Hsp70 transgene before and following the time course after paGFP-Pol II photoactivation. The region of interest is outlined by white dots. (C) Normalized fluorescence intensities of paGFP-Pol II fluorescence decay after photoactivation (FDAP) under the uninduced condition (n = 9). (D) After heat shock, images of mCherry-LacI, paGFP-Pol II (rephotoactivated after heat shock), and merge at the Hsp70 transgene. Error bars indicate SD. Bar, 10 μm.

Figure 2.

Biochemical analysis of steady-state paused Pol II kinetics. (A) Schematic showing the kinetic fates of paused Pol II and short nascent RNA (nasRNA). (k_el) Kinetic constant of paused Pol II elongation; (k_t) kinetic constant of paused Pol II termination; (k_d) short nascent RNA decay constant; ([RNA]_fr,) free Hsp70 short nascent RNA; ([RNA]_ch) chromatin-bound Hsp70 short nascent RNA. (B) The equations used to calculate the pause Pol II elongation and termination rate constants (see the Supplemental Material). (v) Elongation rate; (L) gene length; ([Pol II]_pr) Pol II fraction in pause region; ([Pol II]_gb) Pol II fraction in gene body region. (C) Estimation of k_el from GRO-seq (and PRO-seq) read fractions in Hsp70 (50-bp bins; n = 14 independent data sets). The inset shows read fractions in pause region (−50 to +250 from the transcription start site [TSS]) and gene body (+300 to 2.4 kb). (D) Measuring the ratio between free and chromatin-bound short nascent RNA by qRT–PCR in uninduced (UI; n = 9) and 15-min heat-shock (HS; n = 8) conditions. (E) Estimation of k_d from free Hsp70 short nascent RNA decay after Triptolide (10 μM) addition (qRT–PCR). Each time point is normalized to the pretreatment level. (C–E) Error bars indicate the SEM.

Figure 3.

Optical measurement of the termination rate of paused Pol II at the Hsp70 transgene in living cells. (A) Schematic diagram outlining overall logic of using Flavopiridol to measure paused Pol II termination kinetics. (B) Illustration of the experimental scheme. (C) Semi-log plot of normalized fluorescence intensities of paGFP-Pol II under uninduced condition in control (n = 9) and with Flavopiridol treatment (n = 7). Data are corrected for background Pol II signal (see the Materials and Methods). Error bars indicate SD. (D) Half-lives of paused Pol II decay with Flavopiridol treatment and control. (C,D) Error bars indicate SEM.

Figure 4.

Kinetics of early elongating Pol II at Hsp70 during heat-shock induction. (A) Estimation of initial Pol II escape rate using live-cell imaging at the endogenous Hsp70 upon heat shock. The imaging data from a previous study (Zobeck et al. 2010) were analyzed (n = 33). (B) Contribution of premature termination to Pol II escape rate upon heat-shock induction. (UI) Uninduced condition; (HS) heat-shock induction. The Y-axis is in a nonlinear scale. (C) Model of early elongating Pol II kinetics under uninduced and heat-shock-activated conditions. The width of an arrow reflects the magnitude of the rate.