Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P identified by in vitro selection

Novel RNA substrates for the ribozyme from Bacillus subtilis ribonuclease P (P RNA) have been obtained by in vitro selection. The selection method involves cleavage of a circular RNA library by the P RNA, isolation of the linear cleavage product, and regeneration of circular RNA to allow amplification and multiple cycles of selection. The use of circular RNA ensures that potential substrates can be selected without restricted location of the cleavage site. Such a selection method has been used previously to isolate RNA motifs that undergo autolytic cleavage with Pb2+ [Pan, T., & Uhlenbeck, O. (1992) Biochemistry 31, 3887-3895]. The circular RNA pool after eight cycles of selection was cleaved by the B. subtilis P RNA as efficiently as a pre-tRNA(Phe) substrate, estimated to be more than 10 orders of magnitude better than the unselected RNA library. Kinetic analysis of individual variants showed that the kcat/KM of the selected RNA was up to 4-fold higher than that of the pre-tRNA(Phe). When cleavage was carried out with Escherichia coli P RNA, the selected RNA was 10-60-fold less reactive than the reaction of the pre-tRNA(Phe). Two distinct classes of variants are selected, both of which appear to differ significantly from the known P RNA substrates. Terminal truncation experiments suggest that a large number of nucleotides in the class I variants can be deleted without affecting the cleavage activity. The resulting minimal class I substrates contain a short stem-loop with no other apparent helical structures. The class II substrates are cleaved within a putative helical stem that is formed entirely by the primer sequences.(ABSTRACT TRUNCATED AT 250 WORDS)