Insights into the quality of DnaA boxes and their cooperativity
Insights into the quality of DnaA boxes and their cooperativity
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DOI：
10.1016/j.jmb.2005.10.036
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发表时间：
2006-01-06
影响因子：
5.6
通讯作者：
Atlung, T
中科院分区：
文献类型：
--
作者：
Hansen, FG;Christensen, BB;Atlung, T
携带mioC启动子区及其两个DnaA盒的质粒在滴定DnaA蛋白方面与携带复制失活的oriC区及其五个DnaA盒的质粒一样有效。以各种方式突变mioC启动子上游的两个DnaA盒，以研究DnaA盒之间的协同性，并在体内研究体外定义的9聚体DnaA盒共有序列（TTA/（T）TNCACA）。以两种互补的方式确定DnaA盒的质量和协同性：作为导致dnaA启动子去阻遏的DnaA蛋白的滴定，以及作为由DnaA蛋白结合到DnaA盒引起的mioC启动子的阻遏。DnaA蛋白的滴定与mioC启动子的阻遏相关。正常启动子近端盒（TTTTCCACA）的滴定和阻遏水平强烈依赖于启动子远端位置的DnaA盒的存在和质量，而序列为TTATCCACA的启动子近端DnaA盒滴定DnaA蛋白，并在没有启动子远端DnaA盒的情况下引起mioC启动子的显著阻遏。确定了位于启动子近端位置的八个不同共有DnaA盒的质量：TTATCCACA对DnaA蛋白具有最高的亲和力。在第三位，A优于T，在第五位的四种可能性可以排序为C > A >= G > T。使用纯化的DNA结合结构域的DnaA蛋白的平行体外实验给出了相同的排名的8个DnaA盒的结合亲和力。(c)2005爱思唯尔有限公司保留所有权利。
Plasmids carrying the mioC promoter region with its two DnaA boxes are as efficient in titration of DnaA protein as plasmids carrying a replication-inactivated oriC region with its five DnaA boxes. The two DnaA boxes upstream of the mioC promoter were mutated in various ways to study the cooperativity between the DnaA boxes, and to study in vivo the in vitro-defined 9mer DnaA box consensus sequence (TTA/(T)TNCACA). The quality and cooperativity of the DnaA boxes were determined in two complementary ways: as titration of DnaA protein leading to derepression of the dnaA promoter, and as repression of the mioC promoter caused by the DnaA protein binding to the DnaA boxes. Titration of DnaA protein correlated with repression of the mioC promoter. The level of titration and repression with the normal promoter-proximal box (TTTTCCACA) depends strongly on the presence and the quality of a DnaA box in the promoter-distal position, whereas a promoter-proximal DnaA box with the sequence TTATCCACA titrated DnaA protein and caused significant repression of the mioC promoter without a promoter-distal DnaA box. The quality of the eight different consensus DnaA boxes located in the promoter proximal position was determined: TTATCCACA had the highest affinity for DnaA protein. In the third position, A was better than T, and the four possibilities in the fifth position could be ranked as C > A >= G > T. Parallel in vitro experiments using a purified DNA-binding domain of DnaA protein gave the same ranking of the binding affinities of the eight DnaA boxes. (c) 2005 Elsevier Ltd. All rights reserved.