TFIIF and TFIIS Enhance the Mechanical Persistence of Transcript Elongation by RNA Polymerase II

Mounting evidence suggests that transcript elongation in eukaryotic organisms is regulated via a variety of mechanical cues affecting entry into and exit from transient transcriptional pauses or permanent arrest. A number of accessory factors, including TFIIF and TFIIS, are implicated in the control of these processes. Here, we investigate the interactions of RNA polymerase II (RNAPII) with these factors in a single-molecule optical trapping assay. When monitoring the response of elongation complexes - containing RNAPII and combinations of TFIIF and TFIIS - to controlled mechanical loads, we find that at low force both factors are independently capable of returning arrested RNAPII to productive elongation. At high forces, TFIIF synergistically enhances TFIIS-induced transcriptional re-start but is itself incapable of re-starting transcription. Results obtained with a cleavage-defective TFIIS mutant suggest that TFIIS, in addition to its well-established role in catalyzing transcript cleavage, may relieve arrest via a second, cleavage-independent mechanism. Importantly, the factors' activities do not result in a significantly enhanced stall force, but rather promote the persistence of RNAPII in transcriptionally active states when faced with mechanical obstacles.Our studies also uncover unexpected insights into RNAPII-intrinsic mechanisms underlying transient transcriptional pauses. Both the force-dependence of pause entry and a direct visualization of the process at near-basepair resolution reveal two distinct mechanisms: under forces opposing transcription, pauses typically originate from a backtracking event, whereas under assisting loads a backtrack-independent pause mechanism dominates. Pause duration distributions provide additional mechanistic insights, suggesting that backtracking is not a purely diffusive process, but that instead the extent of backtracking may be restricted by mechanisms intrinsic to RNAPII.Taken together, these results provide a glimpse at how nature resolves obstacles to transcription through the concerted interactions of RNAPII with multiple accessory factors.