Functional reconstitution of an archaeal splicing endonuclease in vitro.
Sulfolobus tokodaii strain 7 is one of archaea 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 these two ORFs lacks a putative catalytic amino acid residue for the nuclease activity. To investigate their functions, the two ORFs were individually expressed in E. coli, partially purified, and tested for their nuclease activities in vitro. Using in vitro transcribed tRNA precursors as substrates, we found that the two ORF products are concurrently required to cleave exon-intron junctions. Our finding implies that the splicing endonuclease of the organism is a multi-subunit complex composed of the two ORF products.
Yoshinari
Shigeo
S
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Fujita
Shinji
S
Masui
Ryoji
R
Kuramitsu
Seiki
S
Yokobori
Shin-ichi
S
Kita
Kiyoshi
K
Watanabe
Yoh-ichi
Y
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012364:RNA, Transfer, Trp / Q000737:chemistry / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D012322:RNA Precursors; D012364:RNA, Transfer, Trp; D004722:Endoribonucleases; C051012:splicing endonuclease