Effects of 5 ' leader and 3 ' trailer structures on pre-tRNA processing bynuclear RNase P

Eukaryotic transfer RNA precursors (pre-tRNAs) contain a 5' leader precedin g the aminoacyl acceptor stem and a 3' trailer extending beyond this stem. An early step in pre-tRNA maturation is removal of the 5' leader by the end oribonuclease, RNase P. Extensive pairing between leader and trailer sequen ces has previously been demonstrated to block RNase P cleavage, suggesting that the 5' leader and 3' trailer sequences might need to be separated for the substrate to be recognized by the eukaryotic holoenzyme. To address whe ther the nuclear RNase P holoenzyme recognizes the 5' leader and 3' trailer sequences independently, interactions of RNase P with pre-tRNA(Tyr) contai ning either the 5' leader, the 3' trailer, or both were examined. Kinetic a nalysis revealed little effect of the 3' trailer or a long 5' leader on the catalytic rate (k(cat)) for cleavage using the various pre-tRNA derivative s. However, the presence of a 3' trailer that pairs with the 5' leader incr eases the K-m of pre-tRNA slightly, in agreement with previous results. Sim ilarly, competition studies demonstrate that removal of a complementary 3' trailer lowers the apparent K-I, consistent with the structure between thes e two sequences interfering with their interaction with the enzyme. Deletio n of both the 5' and 3' extensions to give mature termini resulted in the l east effective competitor. Further studies showed that the nuclear holoenzy me, but not the B. subtilis holoenzyme, had a high affinity for single-stra nded RNA in the absence of attached tRNA structure. The data suggest that y east nuclear RNase P contains a minimum of two binding sites involved in su bstrate recognition, one that interacts with tRNA and one that interacts wi th the 3' trailer. Furthermore, base pairing between the 5' leader and 3' t railer hinders recognition.