Selection of cleavage site by mammalian tRNA 3 ' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' traile rs from pre-tRNAs by cleaving the RNA immediately downstream of the discrim inator nucleotide. Although 3' tRNase can recognize and cleave any target R NA that forms a pre-tRNA-like complex with another RNA, in some cases cleav age occurs at multiple sites near the discriminator. We investigated what f eatures of pre-tRNA determine the cleavage site using various pre-tRNA(Arg) variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we con structed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor ste m base-pairs were cleaved one and two nucleotides and two and three nucleot ides, respectively, downstream of the discriminator. On the other hand, pre -tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucle otides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-p air T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleav ed after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp ( N is less than 12) were cleaved 12 - N and 13 - N nt downstream of the disc riminator. Cleavage efficiency of the variants decreased depending on the d egree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are imp ortant for recognition and cleavage by 3' tRNase; (C) 1999 Academic Press.