The N-terminal region is important for the nuclease activity and thermostability of the flap endonuclease-1 from Sulfolobus tokodaii.
This paper reports the biochemical properties of two types of recombinant flap endonuclease-1 (FEN-1) proteins obtained from the thermophilic crenarchaeon, Sulfolobus tokodaii strain 7. One of the two FEN-1 proteins is a product of the gene with AUG as the translational start codon (StoS-FEN-1), which is originally assigned in the database. The other is a product of the gene with a new AUG start codon (StoL-FEN-1), which is inserted at 153 bases upstream of the original AUG codon. Although StoL-FEN-1 showed activity and thermostability, StoS-FEN-1 showed neither activity nor thermostability. The N-terminal region in StoL-FEN-1 was also conserved in all of the FEN-1 homologs deduced from genes from newly isolated Sulfolobus spp. These results strongly suggest that the actual start codon of the fen-1 gene from S. tokodaii is not the originally assigned AUG, but rather is located at about 100 bases upstream of this codon.
Horie
Masanori
M
Preventure Program, Office of Technology Transfer, Japan Science and Technology Agency, Tokyo, Japan. masa-horie@aist.go.jp
Fukui
Kôichi
K
Xie
Minjue
M
Kageyama
Yoshitaka
Y
Hamada
Kazuo
K
Sakihama
Yuri
Y
Sugimori
Kenji
K
Matsumoto
Kazuko
K
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D030541:Databases, Genetic; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D045585:Flap Endonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013822:Thermoplasma / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D045585:Flap Endonucleases