Biochemistry (Washington) 2013-09-24

Discrimination of RNA from DNA by polynucleotide phosphorylase.

Mihaela-Carmen Unciuleac, Stewart Shuman

Index: Biochemistry 52(38) , 6702-11, (2013)

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Abstract

Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also thought to participate in bacterial DNA transactions. Here we used chimeric polynucleotides, composed of alternating RNA and DNA tracts, to analyze whether and how Mycobacterium smegmatis PNPase discriminates RNA from DNA during the 3'-phosphorolysis reaction. We find that a kinetic block to 3'-phosphorolysis of a DNA tract within an RNA polynucleotide is exerted when resection has progressed to the point that a 3'-monoribonucleotide flanks the impeding DNA segment. The position of the pause one nucleotide before the first deoxynucleotide encountered is independent of DNA tract length. However, the duration of the pause is affected by DNA tract length, being transient for a single deoxynucleotide and durable when two or more consecutive deoxynucleotides are encountered. Substituting manganese for magnesium as the metal cofactor allows PNPase to "nibble" into the DNA tract. A 3'-phosphate group prevents RNA phosphorolysis when the metal cofactor is magnesium. With manganese, PNPase can resect an RNA 3'-phosphate end, albeit 80-fold slower than a 3'-OH. We discuss the findings in light of the available structures of PNPase and the archaeal exosome·RNA·phosphate complex and propose a model for catalysis whereby the metal cofactor interacts with the scissile phosphodiester and the penultimate ribose.