title: RNA splicing ligase activity in the archaeon Haloferax volcanii.
abstract: At least two separate enzymes, an endonuclease and a ligase, appear to be involved in tRNA splicing in halophilic archaea. We have identified and partially characterized a splicing ligase activity in cell extracts of Haloferax volcanii that can ligate deproteinized exon products generated in a separate endonuclease reaction. As in vitro transcribed partial intron-deleted derivative of H. volcanii elongator tRNA(Met) is used as substrate for the endonuclease. The ligase can also join the two exons that are independently eluted from the gels. This ligase activity is observed at a range (50 mM to 2.8 M) of monovalent cations in the assays, but is abolished when the enzyme preparations are depleted of the monovalent cations. In contrast, H. volcanii splicing endonuclease has been reported to require divalent cations and is inhibited by monovalent cations. Our endonuclease assays confirm these reports, and also show that the endonuclease is not permanently inactivated even in high monovalent cation containing extracts. The ligase activity in the extracts does not appear to require any divalent cation or exogenously added source of energy or phosphate.
authors.affiliation: Southern Illinois University, Carbondale, IL 62901-4413, USA.
authors.forename: I
authors.initials: I
authors.lastname: Gomes
authors.forename: R
authors.initials: R
authors.lastname: Gupta
mesh_terms: D001105:Archaea / Q000201:enzymology* / Q000254:growth & development; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004795:Enzyme Stability; D005091:Exons; D007438:Introns; D007700:Kinetics; D008718:Methionine-tRNA Ligase / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009994:Osmolar Concentration; D011130:RNA Ligase (ATP) / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
chemical_list: D009838:Oligodeoxyribonucleotides; D012358:RNA, Transfer, Met; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004722:Endoribonucleases; C051012:splicing endonuclease; D008718:Methionine-tRNA Ligase; C108348:tRNA splicing ligase; D011130:RNA Ligase (ATP)