NUCLEAR PRE-TRANSFER-RNA TERMINAL STRUCTURE AND RNASE-P RECOGNITION

Nuclear pre-tRNA transcripts often contain an extension of the aminoac yl stem formed by base pairing between the 5'-leader and 3'-trailing s equences, but the -1 position preceding the mature 5' end is usually l eft unpaired. Considering recently proposed tertiary structural models for RNase P RNAs, we hypothesize that the -1 mismatch prevents a stro ng, coaxially extended aminoacyl stem, which might otherwise stericall y interfere with substrate positioning in the RNase P active site. Thi s hypothesis is tested by creating uninterrupted aminoacyl stem extens ions in four nuclear tRNA precursors that normally have a mismatched n ucleotide at position -1, and comparing their cleavage rates with thos e of the normal precursors. Determinations of K-m and k(cat) values fo r a normal and an altered pre-tRNA(SUP53), which exhibits the most sub tle structural alteration immediately upstream of the cleavage site, i ndicate that the mismatch at position -1 is an important structural re quirement for both substrate affinity and efficient catalysis (and/or product release) by nuclear RNase P. This conclusion is further suppor ted in vivo, where the pre-tRNA(SUP53) mutant precursor lacking the -1 mismatch is shown to accumulate.