The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNAHis structure and end-processing

Abstract

End-maturation reactions, in which the 5′ end leader and 3′ end trailer of precursor tRNA are removed by RNase P and 3′-tRNase, respectively, are early, essential steps in eukaryotic precursor tRNA processing. End-processing enzymes may be expected to contact the acceptor stem of tRNA due to its proximity to both cleavage sites. We constructed matrices of pair-wise substitutions in mid-acceptor stem at nt 3/70 and 4/69 of Drosophila tRNAHis and analyzed their ability to be processed by Drosophila RNase P and 3′-tRNase. In accord with our earlier study of D/T loop processing matrices, we find that tRNA end processing enzymes respond to sequence changes differently. More processing defects were observed with 3′-tRNase than with RNase P, and substitutions at 4/69 reduced processing more than those at 3/70. We evaluated tRNA folding using structure probing nucleases and investigated the contribution of KM and VMax to the processing efficiency of selected variants. In one substitution (C3A), mis-folding correlates with processing defects. In another (C69A), a disruption of structure appears to be transmitted laterally to both ends of the acceptor stem. Poor processing of C69A by RNase P is due entirely to a reduction in VMax, but for 3′-tRNase, it is due to an increase in KM.