Minimum requirements for substrates of mammalian tRNA 3 ' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes a 3' trai ler after the discriminator nucleotide from precursor tRNA (pre-tRNA), To e lucidate the minimum requirements for 3' tRNase substrates, we tested small pre-tRNA(Arg) substrates lacking the D and anticodon stem-loop domain for cleavage by purified pig 3' tRNase, A small pre-tRNA (R-ATW) composed of an acceptor stem, an extra loop, a T stem-loop domain, a discriminator nucleo tide, and a 3' trailer was cleaved more efficiently than the full-length wi ld type. The catalytic efficiencies of three R-ATW derivatives, which were constructed to destroy the original T stem base pairs, were also higher tha n that of the full-length wild type. Pig 3' tRNase efficiently processed a "minihelix" (R-ATM5) that consists of a T stem-loop domain, an acceptor ste m, a discriminator nucleotide, and a 3' trailer, while the enzyme never cle aved a "microhelix" that is composed of a T loop, an acceptor stem, a discr iminator nucleotide, and a 3' trailer. Five R-ATM5 derivatives that have on e to seven base substitutions in the T loop were all cleaved slightly more efficiently than the full-length wild type and slightly less efficiently th an R-ATM5, A helix ("minihelix Delta 1") one base pair smaller than minihel ices was a good substrate, while small helices containing a continuous 10-b ase pair stem were poor substrates. The cleavage of these three small subst rates occurred after the discriminator and one to three nucleotides downstr eam of the discriminator. From these results, we conclude that minimum subs trates for efficient cleavage by mammalian 3' tRNase are minihelices or min ihelices Delta 1, in which there seem to be no essential bases.