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首页> 外文期刊>Journal of Molecular Biology >Specificity of Mammalian Y-box Binding Protein p50 in Interaction with ss and ds DNA Analyzed with Generic Oligonucleotide Microchip.
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Specificity of Mammalian Y-box Binding Protein p50 in Interaction with ss and ds DNA Analyzed with Generic Oligonucleotide Microchip.

机译：用通用寡核苷酸微芯片分析了哺乳动物Y-box结合蛋白p50在与ss和ds DNA相互作用中的特异性。

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摘要

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.
机译：p50蛋白是Y-box结合转录因子家族的成员，并且是YB-1蛋白的对应物。通用微芯片用于分析p50与单（ss）和双链（ds）寡脱氧核糖核苷酸结合的序列特异性。通用微芯片包含4096个单链十八烷氧基核糖核苷酸，其中所有可能的核心6-聚体（4（6）=​​ 4096）在其3'和5'末端均带有简并核苷酸。将寡核苷酸化学固定在固定在载玻片上的聚丙烯酰胺凝胶垫内。已显示p50与通用微芯片的结合对ss GGGG基序最特异，然后对ss CACC和CATC基序最特异。通用微芯片的富含GC的ds寡核苷酸，尤其是那些包含GGTG / CACC，GATG / CATC和GTGG / CCAC异质基序的寡核苷酸，由于与p50相互作用而最不稳定。凝胶移位电泳显示，与携带富含A，T的6-氨基酸的24-mer oligoA-AAATAT-oligoA相比，该蛋白质与含有富含G的6-mer的24-mer oligoA-TGGGGG-oligoA的结合特异性更高。完全与通过微芯片获得的数据相对应。使用凝胶固定的ss和ds DNA片段对DNA结合蛋白的研究提供了一种独特的可能性，可以检测具有短序列基序的这些蛋白的低亲和力复合物，并评估这些基序在具有长识别位点的序列特异性相互作用中的作用。

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