title: Recombinant RNase Z does not recognize CCA as part of the tRNA and its cleavage efficieny is influenced by acceptor stem length.
abstract: One of the essential maturation steps to yield functional tRNA molecules is the removal of 3'-trailer sequences by RNase Z. After RNase Z cleavage the tRNA nucleotidyl transferase adds the CCA sequence to the tRNA 3'-terminus, thereby generating the mature tRNA. Here we investigated whether a terminal CCA triplet as 3'-trailer or embedded in a longer 3'-trailer influences cleavage site selection by RNase Z using three activities: a recombinant plant RNase Z, a recombinant archaeal RNase Z and an RNase Z active wheat extract. A trailer of only the CCA trinucleotide is left intact by the wheat extract RNase Z but is removed by the recombinant plant and archaeal enzymes. Thus the CCA triplet is not recognized by the RNase Z enzyme itself, but rather requires cofactors still present in the extract. In addition, we investigated the influence of acceptor stem length on cleavage by RNase Z using variants of wild-type tRNATyr. While the wild type and the variant with 8 base pairs in the acceptor stem were processed efficiently by all three activities, variants with shorter and longer acceptor stems were poor substrates or were not cleaved at all.
authors.affiliation: Molekulare Botanik, Universität Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schiffer
authors.forename: Sylvia
authors.initials: S
authors.lastname: Rösch
authors.forename: Anita
authors.initials: A
authors.lastname: Marchfelder
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D031665:Oenothera biennis / Q000201:enzymology / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D019642:RNA, Archaeal / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology / Q000235:genetics
chemical_list: D012322:RNA Precursors; D019642:RNA, Archaeal; D018749:RNA, Plant; D012365:RNA, Transfer, Tyr; D011994:Recombinant Proteins; D012316:RNA Nucleotidyltransferases; C023198:tRNA nucleotidyltransferase; D004722:Endoribonucleases; C110250:RNase Z; C045782:3'-pre-tRNase