Purification and characterization of an endonuclease from Xenopus laevis ovaries which accurately processes the 3' terminus of human pre-tRNA-Met(i) (3' pre-tRNase).
We have previously reported that the primary transcript of the human tRNAMeti gene undergoes accurate processing to a mature 72-nucleotide species by activities present in the high speed supernatant of Xenopus laevis ovarian extracts (Zasloff, M., Santos, T., Romeo, P., and Rosenberg, M. (1982a) J. Biol. Chem. 257, 7857-7863). We now report the purification and characterization of the enzyme which processes the 3' terminus of the human pre-tRNAMeti species. The activity has been purified about 500-fold from a high speed supernatant of X. laevis ovarian extracts by standard methods. It appears to function as a single polypeptide with a molecular weight of about 97,400. The enzyme generates the mature 3' terminus with a single endonucleolytic cut, also yielding the intact 3' trailer. The endonuclease has a striking preference for the 5' processed pre-tRNAMeti, exhibiting little or no activity in vitro on the intact primary transcript. The enzyme acts similarly with the pre-tRNAAla species of Bombyx mori, suggesting that it possesses a broad substrate range. The requirement of the 3' processing endonuclease for a processed 5' terminus suggests that eukaryotic pre-tRNA processing should follow an ordered cutting sequence in vivo with processing of the 5' leader preceding 3' end maturation.
D000818:Animals; D001483:Base Sequence; D004722:Endoribonucleases / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D009694:Nucleic Acid Precursors / Q000032:analysis*; D009841:Oligonucleotides / Q000032:analysis; D010053:Ovary / Q000201:enzymology; D012322:RNA Precursors; D012343:RNA, Transfer / Q000096:biosynthesis / Q000235:genetics*; D012346:RNA, Transfer, Amino Acyl / Q000032:analysis*; D012358:RNA, Transfer, Met; D014158:Transcription, Genetic; D014982:Xenopus laevis / Q000378:metabolism*
D009694:Nucleic Acid Precursors; D009841:Oligonucleotides; D012322:RNA Precursors; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C045783:pre-tRNA(Met); D012343:RNA, Transfer; D004722:Endoribonucleases; C045782:3'-pre-tRNase