Carruthers, A. | Search bioRxiv

bioRxiv2025

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

Nucleotidyltransferases (NTases) of Pol{beta} superfamily 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 Pol{beta}). Here we report comprehensive characterization of two RlaP from Pseudomonas fluorescens and Pseudomonas aeruginosa (PfRlaP and PaRlaP), respectively. Our study showed that, among macromolecules isolated from E. coli cells, only RNAs are the substrates of RlaP. In vitro 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 PfRlaP 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 in vivo scenario where cell survival requires the involvement of RlaP in RNA repair to restore the biological function of the damaged RNAs.

biochemistry↗

bioRxiv2021

Sequential rescue and repair of stalled and damaged ribosome by bacterial PrfH and RtcB

In bacteria, rescue of stalled ribosomes due to 3-truncated mRNAs is carried out by the ubiquitous trans-translation system as well as alternative ribosome-rescue factors such as ArfA and ArfB. It is unclear, however, how the stalled ribosomes caused by ribosomal damages are rescued. Here, we report that a bacterial system composed of PrfH and RtcB not only rescues a stalled ribosome resulting from a specific damage in the decoding center but also repairs the damage afterwards. Peptide release assays reveal that PrfH is only active with the damaged ribosome, but not with the intact one. A 2.55-angstrom cryo-EM structure of PrfH in complex with the damaged 70S ribosome provides molecular insight into specific recognition of the damage site by PrfH. RNA repair assays demonstrate that PrfH-coupled RtcB efficiently repairs the damaged 30S ribosomal subunit, but not the damaged tRNAs. Thus, our studies have uncovered a biological operation by a pair of bacterial enzymes, aiming to reverse the potentially lethal damage inflicted by an invading ribotoxin for cell survival.

biochemistry↗