Transcription factors IIS and IIF enhance transcription efficiency by differentially modifying RNA polymerase pausing dynamics

Abstract

Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to stimulate transcription elongation. Here, we use a single-molecule transcription elongation assay to study the effects of both factors. We find that these transcription factors enhance overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observe that both factors enhance the processivity of RNA polymerase II through the nucleosomal barrier. The effects of TFIIS and TFIIF are quantitatively described using the linear Brownian ratchet kinetic model for transcription elongation and the backtracking model for transcriptional pauses, modified to account for the effects of the transcription factors. Our findings help elucidate the molecular mechanisms by which transcription factors modulate gene expression.

Keywords: Pol II; enzyme kinetics; optical tweezers; yeast.

Fig. 1.

Single-molecule transcription elongation in the presence of transcription factors TFIIS and TFIIF. (A) Experimental geometry for the single-molecule transcription assay in opposing force configuration. (B) Example traces of Pol II transcription in the absence of factors (black), with TFIIS (blue), with TFIIF (red), and with TFIIF/TFIIS (green). (C) An enlarged typical example trace of Pol II transcription with TFIIS (Left) and TFIIF (Right) at high opposing force (∼10 pN). (D) Pause-free velocity against applied forces. Error bars represent SEMs. Note that we excluded the apparent pause density of Pol II in the 7-pN to 10-pN range in the plot because the mean stall force of Pol II is about 7 pN. (E) Mean pause densities of Pol II in the absence and presence of transcription factors within the force range studied. Vertical lines represent SEMs. (F) The cumulative pause duration distributions of Pol II (black line) and Pol II with TFIIS (blue line) in the 4-pN to 7-pN opposing force range. Dashed lines represent fits from the models (Fig. 3). (G) The cumulative pause duration distributions of Pol II (black line), Pol II with TFIIF (red line), and Pol II with TFIIF/TFIIS (green line) in the 4-pN to 7-pN opposing force range.

Fig. 2.

Transcription factors TFIIF and TFIIS enhance Pol II elongation through the nucleosome. (A) Example traces of Pol II transcription on nucleosomal DNA in the absence of factors (black), with TFIIF (red), with TFIIS (blue), and with TFIIF/TFIIS (green). Some molecules stop in the nucleosome region (right side). (B) Mean dwell times against distance from the nucleosomal dyad. Error bars represent SEMs. (C) Histograms of transcription arrest sites in the presence of different factors. Numbers are the percentage of Pol II molecules that passed through the nucleosome. The extended NPS region (−115 nt to +85 nt) is highlighted in yellow. All nucleosomal transcription experiments were done under an assisting load (Methods).

Fig. 3.

Kinetic model of transcription elongation by Pol II in the presence of TFIIS. Transcription elongation by Pol II is composed of the on-pathway elongation (green) and the off-pathway pausing (purple). Forward translocation (k₁) competes with entry into backtracked pauses (k_b1). In the absence of TFIIS, pause recovery requires forward diffusion of the enzyme (k_f) to a pretranslocated state such as TEC_n,0. TFIIS introduces a new pause recovery mechanism (k_r, red arrows) that takes a backtracked Pol II in the state TEC_i,−j to the on-pathway posttranslocated state TEC_i−j−1,1. Cartoon configurations of Pol II TECs in the pre- and posttranslocated and 1-bp backtracked states show that TFIIS-stimulated transcript cleavage rescues the 1-bp backtracked Pol II complex (TEC_n,−1), transferring it to the elongation-competent posttranslocated state TEC_n−2,1. The purple arrow represents the active site of the enzyme. The RNA transcript and template DNA are shown in red and blue, respectively. N represents NTP.