Functional reconstitution of a crenarchaeal splicing endonuclease in vitro.
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.
Yoshinari
Shigeo
S
Department of Biomedical Chemistry, Graduate School of Medicine, The University of Tokyo, 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
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*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D004706:Endodeoxyribonucleases; C064668:endA protein, bacteria