Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene.
The 565 amino-acid PRIB protein with a Zn(II)2Cys6 zinc-cluster DNA-binding motif is the expression product of the priB gene, which is most actively transcribed in an early stage of fruiting-body formation by the basidiomycete, Lentinus edodes. PRIB produced in Escherichia coli using the bacteriophage T7 expression system was purified by ion-exchange chromatographies and then subjected to random binding-site selection analysis using a pool of random 24-bp oligonucleotides with 13-bp PCR primer sites at each end. The oligonucleotides (50 bp) selected for PRIB binding were cloned into pUC19. A total of 303 cloned DNA fragments were picked randomly and sequenced. The PRIB binding sites could be grouped into 25 individual sequences, suggesting a consensus sequence of 16 bp, 5' GGGGGGGACAGGANCC 3'. Gel mobility-shift assaying of 10 randomly selected sequences all revealed a reasonable band shift. DNase I footprinting analysis of the 50-bp DNA fragment containing the sequence most similar to the consensus sequence showed that PRIB protects the entire 16-bp sequence from digestion by DNase I.
Miyazaki
Y
Y
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute Technology, Yokohama.
Tsunoka
O
O
Shishido
K
K
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011124:Polyporaceae / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000502:physiology*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C086004:PriB protein, E coli; D003545:Cysteine