| The regulation of RNA polymerase directly controls mRNA levels in the cell and represents a central control point for cellular function and behavior. In eukaryotes, RNA polymerase II (RNAP II) transcribes in the presence of structural barriers such as nucleosomes. Under these circumstances, one strategy to allow efficient elongation is to modify the template, i.e. to remodel or remove the barriers to transcription. An alternative strategy is to modify the polymerase, i.e. to increase the ability of the molecular motor to overcome such barriers. In this context, we sought to determine the range of opposing forces in which yeast RNAP II can efficiently operate in both the absence and presence of TFIIS, a transcription elongation factor known to increase transcription through nucleosomes. By applying a single-molecule dual-trap optical-tweezer assay combined with a novel method to enrich for active complexes we found that the response of RNAP II to force is entirely characterized by effects on backtracking. The enzyme ceased transcription at 7.5 +- 2 pN where enzymes were no longer able to recover from backtracks. Increasing the rate of backtrack recovery via the addition of TFIIS allowed transcription to proceed up to a force of 16.9 +- 3.4 pN where enzymes backtrack so frequently that net RNA chain elongation ceased even though transcription velocity was unaffected. The ability of TFIIS to increase the functional force range of RNAP II is a novel consequence of its known biochemical properties and demonstrates the general capability of a regulatory factor to allow a motor to operate against increased loads. |
|