Direct observation of processive exoribonuclease motion using optical tweezers

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.

Development of a 3-DOF Mobile Positioning Mechanism with 6 Contact Points

In this paper, we describe the design, development and experimental results of a 3-DOF precise inchworm mechanism with six contact points. During the last ten years, we have developed an omnidirectional and holonomic inchworm mechanism to provide flexible, compact, and precise microscopic processing. In a previous mechanism, four piezoelectric actuators connected a pair of U-shaped electromagnets arranged to cross each other so that the mechanism can move precisely in any direction. However, positioning repeatability was made difficult by an inclination of the U-shaped electromagnets. Therefore, we designed a new omnidirectional inchworm mechanism composed of a pair of Y-shaped electromagnets and six piezoelectric actuators to prevent the inclination of the electromagnets. In addition, we explain the drive principle of the newly developed mechanism. Finally, we show the experimental results of the positioning repeatability of translational motions in four directions with a payload.