Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene.
Determination of the DNA-binding sequences of the Zn(II)2Cys6 zinc-cluster-containing PRIB protein, derived from the basidiomycete Lentinus edodes gene.
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测定源自担子菌香菇基因的 Zn(II)2Cys6 锌簇 PRIB 蛋白的 DNA 结合序列。
DOI：
10.1093/oxfordjournals.jbchem.a021866
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
K. Shishido
中科院分区：
文献类型：
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作者：
Y. Miyazaki;O. Tsunoka;K. Shishido
具有 Zn(II)2Cys6 锌簇 DNA 结合基序的 565 个氨基酸 PRIB 蛋白是 priB 基因的表达产物，该基因在担子菌、香菇的子实体形成的早期阶段转录最活跃。使用噬菌体 T7 表达系统在大肠杆菌中产生的 PRIB 通过离子交换色谱进行纯化，然后使用两端各有 13 bp PCR 引物位点的随机 24 bp 寡核苷酸库进行随机结合位点选择分析。将选择用于 PRIB 结合的寡核苷酸（50 bp）克隆到 pUC19 中。随机挑选总共303个克隆DNA片段并进行测序。 PRIB 结合位点可分为 25 个单独的序列，表明共有序列为 16 bp，5' GGGGGGGACAGGANCC 3'。 10 个随机选择的序列的凝胶迁移率位移测定均显示出合理的带位移。对包含与共有序列最相似的序列的 50 bp DNA 片段进行 DNase I 足迹分析表明，PRIB 可以保护整个 16 bp 序列免遭 DNase I 消化。
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.