The effects of matrices of paired substitutions in mid-acceptor stem on Drosophila tRNA(His) structure and end-processing.
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 tRNA(His) 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 K(M) and V(Max) 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 V(Max), but for 3'-tRNase, it is due to an increase in K(M).
Mohan
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Natural Sciences/Biology, York College of The City University of New York, New York, Jamaica, 11451, USA.
Levinger
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D000818:Animals; D001482:Base Composition; D020029:Base Pairing / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004331:Drosophila melanogaster / Q000166:cytology / Q000201:enzymology / Q000235:genetics*; D004722:Endoribonucleases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D023282:Nuclease Protection Assays; D012323:RNA Processing, Post-Transcriptional; D016337:RNA, Catalytic / Q000378:metabolism; D012354:RNA, Transfer, His / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P; D006163:Ribonuclease T1 / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism
D016337:RNA, Catalytic; D012354:RNA, Transfer, His; D004722:Endoribonucleases; D043262:Ribonuclease P; C045782:3'-pre-tRNase; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic