期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：49
页码：15101-15106
DOI：10.1073/pnas.1514028112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceBacteria regulate the synthesis and degradation of RNA molecules to ensure timely and robust responses to an ever-changing environment. The transcripts lifetime can be influenced profoundly by a secondary structure that can form in the RNA and that may inhibit or promote its digestion by RNases. The molecular mechanisms by which RNases interact with structured RNAs therefore are of great interest. In this study we used optical tweezers to investigate the mechanistic properties of two such enzymes from Escherichia coli, polynucleotide phosphorylase and RNase R. Our results offer new insights into the functional characteristics of these two enzymes, including the sequence-dependent behavior of RNase R and the presence of discrete steps of six or seven nucleotides taken by polynucleotide phosphorylase. 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.
