DEFINING THE SEQUENCE SPECIFICITY OF THE SACCHAROMYCES-CEREVISIAE DNA-BINDING PROTEIN REB1P BY SELECTING BINDING-SITES FROM RANDOM-SEQUENCE OLIGONUCLEOTIDES
DEFINING THE SEQUENCE SPECIFICITY OF THE SACCHAROMYCES-CEREVISIAE DNA-BINDING PROTEIN REB1P BY SELECTING BINDING-SITES FROM RANDOM-SEQUENCE OLIGONUCLEOTIDES
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DOI：
10.1002/yea.320100608
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发表时间：
1994-06-01
期刊：
影响因子：
2.6
通讯作者：
BRANDL, CJ
中科院分区：
文献类型：
--
作者：
LIAW, PCY;BRANDL, CJ
我们已经使用了随机选择协议来定义的共识和范围的结合位点的酿酒酵母REB 1蛋白。在不保留超过99.9%的随机序列的条件下，对特异性结合到与谷胱甘肽-琼脂糖凝胶偶联的GST-REB 1 p融合蛋白的35个元件进行测序。其中22个元素含有核心序列CGGGTRR，除了一个元素外，其余所有元素都只含有一个与核心的偏差。在核心序列中，唯一绝对保守的残基是三个连续的G残基。核苷酸使用矩阵的统计分析表明，REB 1 p结合位点也延伸到侧翼序列，REB 1 p结合的最佳序列是GNGCCGGGGTAACNC。有一个积极的相关性之间的能力的网站，以结合在体外和激活转录在体内，然而，存在的不符合表明，结合位点可能有助于更多的转录激活比简单地允许蛋白质结合。有趣的是，其中一个REB 1 p结合元件的DNA酶1足迹明显长于具有相似亲和力的其他元件。对其序列的分析表明，在相对链上可能存在第二个REB 1 p结合位点。这表明，两个位置接近的低亲和力位点可以作为一个高活性位点一起发挥作用。此外，用随机定义的REB 1 p结合位点进行的数据库搜索表明，相关元件通常存在于“TATA-少”启动子中。
We have used a random selection protocol to define the consensus and range of binding sites for the Saccharomyces cerevisiae REB1 protein. Thirty-five elements were sequenced which bound specifically to a GST-REB1p fusion protein coupled to glutathione-Sepharose under conditions in which more than 99.9% of the random sequences were not retained. Twenty-two of the elements contained the core sequence CGGGTRR, with all but one of the remaining elements containing only one deviation from the core. Of the core sequence, the only residues that were absolutely conserved were the three consecutive G residues. Statistical analysis of a nucleotide-use matrix suggested that the REB1p binding site also extends into flanking sequences with the optimal sequence for REB1p binding being GNGCCGGGGTAACNC. There was a positive correlation between the ability of the sites to bind in vitro and activate transcription in vivo; however, the presence of non-conformants suggests that the binding site may contribute more to transcriptional activation than simply allowing protein binding. Interestingly, one of the REB1p binding elements had a DNAse 1 footprint appreciably longer than other elements with similar affinity. Analysis of its sequence indicated the potential for a second REB1p binding site on the opposite strand. This suggests that two closely positioned low-affinity sites can function together as a highly active site. In addition, database searches with some of the randomly defined REB1p binding sites suggest that related elements are commonly found within 'TATA-less' promoters.