Schematic summary of the E. coli A49 second-site suppressor mutations isolated from this study. (A) Among other roles, RNase P is responsible for the 5′-end maturation of tRNAs by cleaving the 5′ leader sequences found on pre-tRNA transcripts. In E. coli strains carrying the wild-type rnpA allele, the C5 protein and M1 RNA subunits readily assemble to form the functional holoenzyme. (B) (i) The rnpA49 mutant allele encodes a ts mutation that compromises the assembly and stability of the RNase P holoenzyme at nonpermissive temperatures. This leads to an accumulation of pre-tRNAs with unprocessed 5′-ends, which are often polyadenylated by PAP I and targeted for degradation. (ii) The rnpA49 ts phenotype can be partially suppressed by increasing the gene copy number of either subunit of the mutant RNase P complex (rnpA49 or rnpB) via large genome amplifications, which shifts the equilibrium of assembly to favor the formation of the mutant RNase P holoenzyme. (iii) Rescue of the rnpA49 ts phenotype can also occur via the acquisition of loss-of-function mutations in RNase R. RNase R targets polyadenylated RNAs for degradation, thus inactivation of RNase R likely stabilizes the unprocessed, polyadenylated pre-tRNAs for aminoacylation and/or may also contribute to increased M1 RNA stability and improved assembly of the mutant RNase P holoenzyme. (iv) Lastly, rnpA49 suppressor mutations inactivating Lon protease increase C5A49 protein abundance to promote mutant RNase P holoenzyme assembly and stability.
