基于功能的大肠杆菌RNA聚合酶σ⁷⁰启动子碱基对多态性的筛选与鉴定
Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70).
作者信息
Xu J, McCabe B C, Koudelka G B
机构信息
Department of Biological Sciences, State University of New York at Buffalo, 14260-1300, USA.
出版信息
J Bacteriol. 2001 May;183(9):2866-73. doi: 10.1128/JB.183.9.2866-2873.2001.
Abstract
We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.
摘要
我们进行了两组体外筛选实验，以剖析 -10 碱基序列在决定大肠杆菌 RNA 聚合酶-σ⁷⁰ 与启动子形成稳定复合物的速率和效率方面的作用。我们鉴定出了两类序列：（i）能与 RNA 聚合酶迅速形成抗肝素复合物的序列，或（ii）在极低 RNA 聚合酶浓度下形成抗肝素复合物的序列。在这两种条件下筛选出的序列彼此不同，且与共有 -10 序列也不同。所筛选出的启动子具有预期增强的结合和动力学特性，并且在体外指导 RNA 合成方面，其功能比共有启动子序列更好。对所筛选启动子功能的详细分析表明，这条多步骤途径中的每一步可能都有不同的序列要求，这意味着强启动子的序列并非包含每一步的最佳序列，而是一个折衷序列，能使所有步骤在最小限制下进行。
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