Nuclear pre-tRNA terminal structure and RNase P recognition

Nuclear pre-tRNA transcripts often contain an extension of the aminoacyl stem formed by base pairing between the 5'-leader and 3'-trailing sequences, but the -1 position preceding the mature 5' end is usually left unpaired. Considering recently proposed tertiary structural models for RNase P RNAs, we hypothesize that the -1 mismatch prevents a strong, coaxially extended aminoacyl stem, which might otherwise sterically interfere with substrate positioning in the RNase P active site. This hypothesis is tested by creating uninterrupted aminoacyl stem extensions in four nuclear tRNA precursors that normally have a mismatched nucleotide at position -1, and comparing their cleavage rates with those of the normal precursors. Determinations of Km and kcat values for a normal and an altered pre-tRNA(SUP53), which exhibits the most subtle structural alteration immediately upstream of the cleavage site, indicate that the mismatch at position -1 is an important structural requirement for both substrate affinity and efficient catalysis (and/or product release) by nuclear RNase P. This conclusion is further supported in vivo, where the pre-tRNA(SUP53) mutant precursor lacking the -1 mismatch is shown to accumulate.