Substrate and enzyme determinants for recognition by human mitochondrial RNase P

RNase P enzymes of widely varying architectures recognize the 5'-leader/acceptor-stem junction and the D/T loop-interaction region of precursor tRNAs to direct cleavage to the 5' end of tRNAs. In contrast, human mitochondrial RNase P (mtRNase P) encases the entire tRNA with the aid of the methyltransferase subcomplex TRMT10C-SDR5C1. Here, we performed a kinetic analysis of substrate recognition by mtRNase P using substrate and protein variants. Surprisingly, processing by mtRNase P was found to be more efficient for tRNA precursors with longer 5' extensions and decreased sharply at a leader length of 1 nt. MtRNase P also employs a more rigid "measuring mechanism" for cleavage-site selection than the related single-subunit enzymes, so that even substrates with a G:C base-pair extension of the acceptor stem are cleaved predominantly at the canonical site. The specific contacts of TRMT10C-SDR5C1 with the anticodon loop are not crucial for efficient processing, but without interactions with the pre-tRNA, TRMT10C-SDR5C1 is unable to stimulate cleavage by the nuclease subunit PRORP, also explaining why mtRNase P reaches its limits with the D-armless mitochondrial tRNASer(AGY). Our findings set human mtRNase P apart in terms of substrate recognition from all other known forms of RNase P, including the related single-polypeptide PRORPs.