A single-molecule technique to study sequence-dependent transcription pausing

ABSTRACT We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than;100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to;5 bp by using runoff transcription as an un-ambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DtR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or ‘‘backtracks,’ ’ at the DtR2 pause sequence