Direct observation of processive exoribonuclease motion using optical tweezers

• DOI: 10.1073/pnas.1514028112
• Publication Date: 2015-11-24T03:19:00Z
• Authors: Furqan M. Fazal; Daniel J. Koslover; Ben F. Luisi; Steven M. Block
• Keywords: Exoribonuclease; Polynucleotide phosphorylase; Exosome complex; Degradosome; Processivity; RNase PH

Bacterial RNases catalyze the turnover of RNA and are essential for gene expression quality surveillance transcripts. In Escherichia coli, exoribonucleases RNase R polynucleotide phosphorylase (PNPase) play critical roles in degrading RNA. Here, we developed an optical-trapping assay to monitor translocation individual enzymes along RNA-based substrates. Single-molecule records motion reveal be highly processive: one molecule can unwind over 500 bp a structured substrate. However, enzyme progress is interrupted by pausing stalling events that slow degradation sequence-dependent fashion. We found distance traveled PNPase through dependent on A+U content substrate removal its KH S1 RNA-binding domains reduce processivity without affecting velocity. By periodogram analysis single-molecule records, establish takes discrete steps six or seven nucleotides. These findings, combination with previous structural biochemical data, support asymmetric inchworm mechanism motion. The here well suited studies other exonucleases helicases.