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 wh ich contain a three-nucleotide extension at the 5'-terminus of mature tRNA are minimal substrates for ribonuclease P from both Escherichia coli and Ba cillus subtilis. Variants with a cytidine at position -1 are most efficient ly processed whereas the G -1 variant represents a comparatively poor subst rate. 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 dupl ex. This is in qualitative agreement with the frequency of the occurrence o f the corresponding nucleotides at position -1 in natural substrates, which is highest for pyrimidines and least for G. NMR analyses of the correspond ing acceptor stems reveal that the conformation of the nucleotides at posit ion -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 t hat of A -1 is intermediate between high-anti and syn. The riboses of nucle otides -1 are in all cases predominantly 2'-endo puckered.