RNAseq of Escherichia coli with protein-only RNase P substitutions

The family of RNase P endoribonucleases comprises diverse enzyme architectures ranging from complex ribonucleoprotein assemblies to single polypeptides as small as ~23 kDa termed homologs of Aquifex RNase P (HARPs). We demonstrate that the HARPs of two hyperthermophilic bacteria (Aquifex aeolicus and Thermodesulfatator indicus) and one thermophilic archaeon (Methanothermobacter thermoautotrophicus) confer basic viability to Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. RNA-seq, Northern blot and primer extension data revealed decreased processing efficiency of the HARPs in the E. coli host when acting on precursor tRNAs (pre-tRNAs) that can form acceptor stems extended by G:C or C:G base pairs. HARPs are defective in processing of 4.5S RNA transcripts lacking the T domain of tRNAs. Furthermore, our study revealed that an A residue immediately upstream of the cleavage site increases efficiency and accuracy in reactions catalyzed by HARPs. In the case of A. aeolicus that entirely relies on its HARP for RNase P activity, pre-tRNAs evidently coevolved with the new type of protein-only RNase P by avoiding stable acceptor stem extensions and by encoding an A residue upstream of the native cleavage site in 38 of its 44 tRNA transcripts