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Single molecule analysis of yeast Rrp44 exonuclease reveals a spring-loaded mechanism of RNA unwinding

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Abstract

The eukaryotic exosome catalyzes a series of reactions such as RNA processing and decay. Rrp44 is a key catalytic subunit of the yeast exosome complex and enables multi-enzymatic activities, including endoribonuclease, exoribonuclease and duplex unwinding. Its exoribonuclease and unwinding activities are indispensable for the complete degradation of mRNA that forms a variety of secondary structures. It is, however, unknown how the unwinding activity is coordinated with the exonuclease one. We used single-molecule fluorescence techniques to investigate the mechanism of unwinding by Rrp44 in real time. Surprisingly, we found that Rrp44 does not unwind the RNA duplex each time it digests a single nucleotide off the 3’ end of the single stranded RNA tail. Instead, it accumulates elastic energy during multiple steps of RNA digestion and unwinds several basepairs simultaneously. The kinetic analysis of each unwinding step shows that RNA unwinding, not RNA digestion, determines the overall RNA degradation rate. A series of control experiments varying the RNA sequence, salt concentrations and temperature, and the use of hybrid RNA/DNA duplex demonstrate that the unwinding step size is determined by the physical properties of the enzyme itself, not by the duplex stability. Our studies represent the first example of a multiple hierarchy of stepping for an exonuclease.

© 2011 Optical Society of America

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