The pre-tRNA nucleotide base and 2'-hydroxyl at N(-1) contribute to fidelity in tRNA processing by RNase P.
Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage. Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection. We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site. Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways. Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site. Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive. One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold. Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence. Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo. These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.
Zahler
Nathan H
NH
Department of Biochemistry, Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4973, USA.
Sun
Lei
L
Christian
Eric L
EL
Harris
Michael E
ME
D020029:Base Pairing; D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D009711:Nucleotides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*
D009711:Nucleotides; D010710:Phosphates; D012343:RNA, Transfer; D043262:Ribonuclease P; D008274:Magnesium