Correlation between Processing Efficiency for Ribonuclease P Minimal Substrates and Conformation of the Nucleotide −1 at the Cleavage Position

It is demonstrated that acceptor stem duplexes derived from native tRNAs which contain a three-nucleotide extension at the 5‘-terminus of mature tRNA are minimal substrates for ribonuclease P from both Escherichia coli and Bacillus subtilis. Variants with a cytidine at position −1 are most efficiently processed whereas the G −1 variant represents a comparatively poor substrate. An A −1 acceptor stem variant is a slightly better substrate than the G −1 variant though generally distinctly less efficient than the C −1 duplex. This is in qualitative agreement with the frequency of the occurrence of the corresponding nucleotides at position −1 in natural substrates, which is highest for pyrimidines and least for G. NMR analyses of the corresponding acceptor stems reveal that the conformation of the nucleotides at position −1 correlates with the substrate preferences of Ribonuclease P: Whereas C −1 adopts a conformation characterized by a glycosidic angle in the anti range (close to high-anti), the G −1 is clearly in syn conformation, and that of A −1 is intermediate between high-anti and syn. The riboses of nucleotides −1 are in all cases predominantly 2‘-endo puckered.