Determination of the DNA-Binding Sequences of the Zn(II)2Cys6 Zinc-Cluster-Containing PRIB Protein, Derived from the Basidiomycete Lentinus edodes Gene

Abstract

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.