Bacterial/archaeal protein-only RNase P: complementation in Escherichia coli uncovers coevolution of protein-only RNase P and precursor tRNA structures in Aquifex aeolicus

Abstract

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). The HARPs of two hyperthermophilic bacteria (Aquifex aeolicus and Thermodesulfatator indicus) and one thermophilic archaeon (Methanothermobacter thermautotrophicus) restore (although at reduced growth rate) the viability of Escherichia coli cells with a lethal knockdown of its endogenous RNA-based RNase P. Potential causes for retarded growth were analyzed by RNA-seq, northern blot, and primer extension. This revealed inefficient processing by HARPs in the E. coli host, particularly for precursor tRNAs (pre-tRNAs) with acceptor stems extended by one or more G-C base pairs, and also for the non-tRNA substrate pre-4.5S RNA. Yet, E. coli pre-tRNAs fortuitously carrying an A residue immediately upstream of the (canonical or aberrant) cleavage site were processed more efficiently, particularly by the two bacterial HARPs. Follow-up in vitro processing assays using RNase P model substrates confirmed 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 apparently coevolved with the enzyme, as 38 of the 44 tRNA transcripts carry an A residue and none a stable G-C or C-G bp immediately upstream of the native cleavage site, two attributes that are clearly favorable for accurate and efficient catalysis by this class of protein-only RNase P.