Biochemical and structural characterization of the RlaP family nucleotidyltransferase potentially involved in RNA repair

Nucleotidyltransferases (NTases) of Polβsuperfamily are highly diverse and play many important biological functions. However, many families of NTases remain uncharacterized. Because of physical proximity of the genes encoding NTases of PF10127 family to those encoding RNA ligases involved in RNA repair, PF10127 family of NTases was named RlaP (RNA ligase-associating Polβ). Here we report comprehensive characterization of two RlaP from <i>Pseudomonas fluorescens</i> and <i>Pseudomonas aeruginosa</i> (<i>Pf</i>RlaP and <i>Pa</i>RlaP), respectively. Our study showed that, among macromolecules isolated from <i>E. coli</i> cells, only RNAs are the substrates of RlaP. <i>In vitro</i> assays employing synthetic RNAs as substrates demonstrated that RlaP catalyzes addition of one or two nucleotide monophosphate (NMP) to the 3'-hydroxyl group of RNAs, with the damaged RNAs as the preferred substrates. The crystal structure of <i>Pf</i>RlaP provided insight into molecular recognition of RNA substrate and nucleotide triphosphate (NTP) by RlaP. Aminoacylation assays indicate that RlaP is required to restore biological function of the repaired tRNAs that have suffered excessive damage. Based on our studies, we propose an <i>in vivo</i> scenario where cell survival requires the involvement of RlaP in RNA repair to restore the biological function of the damaged RNAs.