Suppression of theE.coli rnpA49conditionally lethal phenotype via different compensatory mutations

Abstract

ABSTRACTRNase P is an essential enzyme found across all domains of life that is responsible for the 5’-end maturation of precursor tRNA transcripts. Since its discovery in the 1970s, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations. InEscherichia coli, the temperature-sensitivernpA49mutation in the protein subunit of RNase P has arguably been one of the most well-studied and commonly used mutations for examining the enzyme’s activityin vivo. Here we report for the first time naturally-occurring temperature-resistant suppressor mutations ofE. colistrains carrying thernpA49allele. We find thatrnpA49strains can partially compensate the temperature-sensitive defect via gene amplifications of either RNase P subunit (rnpA49orrnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insight into the behavior and complementation of thernpA49allelein vivoand provides direction for follow-up studies regarding RNase P regulation and turnover.