Swapping the domains of exoribonucleases RNase II and RNase R: Conferring upon RNase II the ability to degrade ds RNA

RNase II and RNase R are the two E. coli exoribonucleases that belong to the RNase II super family of enzymes. They degrade RNA hydrolytically in the 3′ to 5′ direction in a processive and sequence independent manner. However, while RNase R is capable of degrading structured RNAs, the RNase II activity is impaired by dsRNAs. The final end‐product of these two enzymes is also different, being 4 nt for RNase II and 2 nt for RNase R. RNase II and RNase R share structural properties, including 60% of amino acid sequence similarity and have a similar modular domain organization: two N‐terminal cold shock domains (CSD1 and CSD2), one central RNB catalytic domain, and one C‐terminal S1 domain. We have constructed hybrid proteins by swapping the domains between RNase II and RNase R to determine which are the responsible for the differences observed between RNase R and RNase II. The results obtained show that the S1 and RNB domains from RNase R in an RNase II context allow the degradation of double‐stranded substrates and the appearance of the 2 nt long end‐product. Moreover, the degradation of structured RNAs becomes tail‐independent when the RNB domain from RNase R is no longer associated with the RNA binding domains (CSD and S1) of the genuine protein. Finally, we show that the RNase R C‐terminal Lysine‐rich region is involved in the degradation of double‐stranded substrates in an RNase II context, probably by unwinding the substrate before it enters into the catalytic cavity. Proteins 2011; © 2011 Wiley‐Liss, Inc.