title: Direct observation of processive exoribonuclease motion using optical tweezers.
abstract: Bacterial RNases catalyze the turnover of RNA and are essential for gene expression and quality surveillance of transcripts. In Escherichia coli, the exoribonucleases RNase R and polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor the translocation of individual enzymes along RNA-based substrates. Single-molecule records of motion reveal RNase R to be highly processive: one molecule can unwind over 500 bp of a structured substrate. However, enzyme progress is interrupted by pausing and stalling events that can slow degradation in a sequence-dependent fashion. We found that the distance traveled by PNPase through structured RNA is dependent on the A+U content of the substrate and that removal of its KH and S1 RNA-binding domains can reduce enzyme processivity without affecting the velocity. By a periodogram analysis of single-molecule records, we establish that PNPase takes discrete steps of six or seven nucleotides. These findings, in combination with previous structural and biochemical data, support an asymmetric inchworm mechanism for PNPase motion. The assay developed here for RNase R and PNPase is well suited to studies of other exonucleases and helicases.
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, CA 94305;
authors.forename: Furqan M
authors.initials: FM
authors.lastname: Fazal
authors.affiliation: Biophysics Program, Stanford University, Stanford, CA 94305;
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Koslover
authors.affiliation: Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom;
authors.forename: Ben F
authors.initials: BF
authors.lastname: Luisi
authors.affiliation: Department of Applied Physics, Stanford University, Stanford, CA 94305; Biophysics Program, Stanford University, Stanford, CA 94305; Department of Biology, Stanford University, Stanford, CA 94305 sblock@stanford.edu.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Block
mesh_terms: D004247:DNA / Q000737:chemistry; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D052898:Optical Tweezers; D012313:RNA / Q000737:chemistry
chemical_list: D012313:RNA; D004247:DNA; D005095:Exoribonucleases
keywords: exoribonuclease; optical trap; processivity; single- molecule biophysics; step size