Backtracking by single RNA polymerase molecules observed at near-base-pair resolution.
Escherichia coli RNA polymerase (RNAP) synthesizes RNA with remarkable fidelity in vivo. 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 3' 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 ( approximately 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.
Shaevitz
Joshua W
JW
Department of Physics, Stanford University, Stanford, California 94305, USA.
Abbondanzieri
Elio A
EA
Landick
Robert
R
Block
Steven M
SM
D020029:Base Pairing; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D016296:Mutagenesis; D012329:RNA, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; C079668:GreA protein, E coli; C079667:GreB protein, E coli; D012329:RNA, Bacterial; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D004247:DNA; D012321:DNA-Directed RNA Polymerases