Applied force reveals mechanistic and energetic details of transcription termination.
Transcription termination by bacterial RNA polymerase (RNAP) occurs at sequences coding for a GC-rich RNA hairpin followed by a U-rich tract. We used single-molecule techniques to investigate the mechanism by which three representative terminators (his, t500, and tR2) destabilize the elongation complex (EC). For his and tR2 terminators, loads exerted to bias translocation did not affect termination efficiency (TE). However, the force-dependent kinetics of release and the force-dependent TE of a mutant imply a forward translocation mechanism for the t500 terminator. Tension on isolated U-tracts induced transcript release in a manner consistent with RNA:DNA hybrid shearing. We deduce that different mechanisms, involving hypertranslocation or shearing, operate at terminators with different U-tracts. Tension applied to RNA at terminators suggests that closure of the final 2-3 hairpin bases destabilizes the hybrid and that competing RNA structures modulate TE. We propose a quantitative, energetic model that predicts the behavior for these terminators and mutant variants.
Larson
Matthew H
MH
Biophysics Program, Stanford University, Stanford, CA 94305, USA.
Greenleaf
William J
WJ
Landick
Robert
R
Block
Steven M
SM
D020121:5' Untranslated Regions; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D001435:Bacteriophages / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics; D013728:Terminator Regions, Genetic; D013816:Thermodynamics; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012321:DNA-Directed RNA Polymerases