Yeast RNase P: catalytic activity and substrate binding are separate functions.
During tRNA biosynthesis the 5'-leader sequences in precursor tRNAs are removed by the ribonucleoprotein RNase P, an enzyme whose RNA moiety is required for activity. To clarify some aspects of the enzyme mechanism, we examined substrate binding and product formation with mutant precursor tRNAs. Mutations G-1----A or U-2----C in the Schizosaccharomyces pombe sup3-e tRNASer, which cause mispairing at or near the top of the acceptor stem, prevent the removal of the 5'-leader sequences by Saccharomyces cerevisiae RNase P. Equilibrium binding studies involving specific gel retardation of RNase P-precursor tRNA complexes showed that complexes with wild-type and A-1 and C-2 mutant precursor tRNAs had very similar dissociation constants (average Kd for sup3 = 1.5 +/- 0.2 nM). Thus, the 5'-terminal nucleotides of mature tRNA, on the 3' proximal side of the RNase P cleavage site, affect the enzyme's catalytic function but not substrate binding. The catalytic integrity of the RNA component of RNase P is not essential for binding of tRNA precursors, as demonstrated by gel retardation of micrococcal nuclease-inactivated enzyme. This suggests a possible role for the protein component of the enzyme in substrate binding. Upon restoration of base pairing to the acceptor stem in the A-1 or C-2 mutants, we found that, in addition to a requirement for pairing at these positions, conservation of the wild-type first and second nucleotides of the tRNA was necessary to obtain maximal cleavage by RNase P. This indicates a distinct sequence preference of this enzyme.
Nichols
M
M
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Söll
D
D
Willis
I
I
D001483:Base Sequence; D002384:Catalysis; D004722:Endoribonucleases / Q000378:metabolism*; D011485:Protein Binding; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000378:metabolism*; D043262:Ribonuclease P; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012568:Schizosaccharomyces / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D012343:RNA, Transfer; D004722:Endoribonucleases; D043262:Ribonuclease P