Backtracking by single RNA polymerase molecules observed at near-base-pair resolution

Escherichia coli RNA polymerase ( RNAP) synthesizes RNA with remarkable fidelity in vivo(1). Its low error rate may be achieved by means of a ' proofreading' mechanism comprised of two sequential events. The first event ( backtracking) involves a transcriptionally upstream motion of RNAP through several base pairs, which carries the 30 end of the nascent RNA transcript away from the enzyme active site. The second event ( endonucleolytic cleavage) occurs after a variable delay and results in the scission and release of the most recently incorporated ribonucleotides, freeing up the active site. Here, by combining ultrastable optical trapping apparatus with a novel two- bead assay to monitor transcriptional elongation with near- base- pair precision, we observed backtracking and recovery by single molecules of RNAP. Backtracking events (similar to 5 bp) occurred infrequently at locations throughout the DNA template and were associated with pauses lasting 20 s to > 30 min. Inosine triphosphate increased the frequency of backtracking pauses, whereas the accessory proteins GreA and GreB, which stimulate the cleavage of nascent RNA, decreased the duration of such pauses.