Molecular and structural basis of a subfamily of PrfH rescuing both the damaged and intact ribosomes stalled in translation.

Abstract

In bacteria, mRNAs degradation and ribotoxin-induced RNA damage are two main events that lead to the stalling of protein translation. The ubiquitous trans-translation system and several alternative rescue factors rescue the stalled ribosomes caused by truncated mRNAs lacking the stop codons. On the other hand, protein release factor homolog (PrfH) is the only known factor to rescue the stalled ribosome damaged by ribotoxins. Here, we show that a subfamily of PrfH, exemplified by PrfH from <i>Capnocytophaga gingivalis</i> (<i>Cg</i>PrfH), rescues both types of stalled ribosomes. Biochemical assays demonstrate that <i>Cg</i>PrfH hydrolyzes the peptides attached to P-site transfer RNAs when in complex with both the damaged and intact ribosomes. Cryo-EM structures revealed that <i>Cg</i>PrfH uses distinct regions to recognize two stalled ribosomes to orient the GGQ motif for peptide hydrolysis. Thus, using a combination of bioinformatic, biochemical, and structural characterization, we have uncovered a family of ribosome rescue factors that have dual activities to resolve two distinct stalled protein translation events in bacteria.