Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA.
Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA.
复制标题
物理、离子和结构因素对分枝杆菌噬菌体 L1 阻遏物与其同源操纵基因 DNA 结合的影响。
DOI：
--
复制
发表时间：
2006
期刊：
影响因子：
--
通讯作者：
S. Sau
中科院分区：
文献类型：
--
作者：
T. Ganguly;P. Chanda;Amitava Bandhu;P. Chattoraj;Malabika Das;S. Sau
为了确定影响L1阻遏物与其同源操纵基因DNA结合的因素，已经进行了几次凝胶移位以及生物信息学分析。数据显示，时间、温度、盐和pH值均极大地影响结合。为了在Tris缓冲液中实现L1阻遏物的最佳操纵子结合，估计时间、温度、盐和pH的最低要求分别为1分钟、32 ℃、NaCl（50 mM）和7.9。有趣的是，发现Na+而不是NH 4+、K+或Li+显著增加Cl蛋白的结合活性高于基础水平。Cl-、柠檬酸盐-、乙酸盐-和H2 PO 4-等阴离子不改变L1阻遏物与其操纵子的结合。我们还表明，在框内缺失突变体的L1阻遏物，不携带假定的HTH基序（在其N-末端）未能结合到其同源的运营商DNA，即使在非常高的浓度。推测的HTH基序被发现高度保守，进化上非常接近Y的调节蛋白。pestis，H. marismortui、黑腹拟步行虫A.我们推测L1阻遏蛋白的N端含有一个HTH基序。进一步分析的推定的二级结构的分枝杆菌噬菌体阻遏物揭示，两个共同的区域，包括超过90%的一级序列存在于所有四个阻遏物分子在这里研究。结果表明，这些共同的区域用于执行相同的功能。
To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.