Analysis of a putative nuclease associated with the Argonaute protein from Rhodobacter sphaeroides

Abstract

The Argonaute protein from the alphaproteobacterium Rhodobacter sphaeroides (RsAgo) binds small guide RNAs to recognize complementary DNA targets. In bacterial cells, RsAgo is bound to both small RNAs and small DNAs, with a preference for foreign genetic elements. RsAgo also promotes degradation of plasmid DNA in vivo. However, RsAgo itself lacks the slicer activity, suggesting the involvement of additional nucleases. To shed light on the molecular mechanism of target processing, we study a putative nuclease which is located in the same operon with RsAgo in the R. sphaeroides genome. This protein has motifs characteristic for the PD(D/E)XK superfamily of nucleases. Numerous attempts to express the wildtype nuclease gene in Escherichia coli were not successful due to a low level of expression and low solubility of the recombinant protein. A codon-optimized sequence of the nuclease gene was cloned and sucessfully overexpressed in E. coli. Different expression vectors were used to produce histidine-tagged nuclease and a fusion protein with a chitin-binding domain, which allowed to obtain highly pure nuclease preparations. Initial assays did not reveal nuclease activity in these proteins. However, it was shown that coexpression of nuclease with the RsAgo protein in E. coli significantly increases the yield of RsAgo and changes the spectrum of genes preferentially targeted by RsAgo. We plan to further determine the role of nuclease in DNA processing in bacterial cells and test its interactions with RsAgo in vitro and in vivo.