Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase.
Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others. Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved. Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules. We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest. Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically.
The Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA.
Nancy R
NR
Forde
David
D
Izhaky
Glenna R
GR
Woodcock
Gijs J L
GJ
Wuite
Carlos
C
Bustamante
D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases