title: Functional reconstitution of a crenarchaeal splicing endonuclease in vitro.
abstract: Sulfolobus tokodaii strain 7 is one of Crenarchaea whose entire genome has been sequenced. The genome sequence revealed that it possesses two open reading frames (ORFs) that are homologous to EndA, a protein responsible for splicing endonuclease activity in Archaea. Interestingly, one of the two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORF products were individually expressed in Escherichia coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursor as a substrate, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease for the organism is a multi-subunit complex composed of the two endA gene products.
authors.affiliation: Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yoshinari
authors.forename: Shinji
authors.initials: S
authors.lastname: Fujita
authors.forename: Ryoji
authors.initials: R
authors.lastname: Masui
authors.forename: Seiki
authors.initials: S
authors.lastname: Kuramitsu
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Yokobori
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Kita
authors.forename: Yoh-ichi
authors.initials: Y
authors.lastname: Watanabe
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002874:Chromosome Mapping; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D019154:Protein Splicing / Q000235:genetics*; D013329:Structure-Activity Relationship; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D008565:Membrane Proteins; D004706:Endodeoxyribonucleases; C064668:endA protein, bacteria