Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA.
Identification of Preferred DNA-Binding Sites for the Thermus thermophilus Transcriptional Regulator SbtR by the Combinatorial Approach REPSA.
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DOI：
10.1371/journal.pone.0159408
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Xie Y
中科院分区：
文献类型：
--
作者：
Van Dyke MW;Beyer MD;Clay E;Hiam KJ;McMurry JL;Xie Y
阐明假定的转录调节因子的生物学功能的第一步之一是确定其优选的DNA结合序列。这可以通过应用组合方法快速有效地实现，所述组合方法涉及非常大量的随机化寡核苷酸和重复选择和扩增步骤以富集高亲和力核酸结合序列。此前，我们开发了新型组合方法限制性内切酶保护、选择和扩增（REPSA），该方法不依赖于配体-核酸复合物的物理分离，而是基于配体依赖性抑制酶模板失活（特别是切割）进行选择IIS型限制性内切酶。因此，REPSA不需要配体的先验知识，使其更适合于发现目的。在这里，我们描述了使用REPSA，大规模平行测序，生物信息学，以确定转录调节SbtR，编码的TTHA 0167基因的模型极端嗜热栖热菌HB8的首选DNA结合位点。从由此产生的位置权重矩阵，我们可以确定多个操纵子可能受到SbtR的调节，并假定这种蛋白质在调节细胞外转运过程中的生物学作用。我们的研究提供了一个概念验证的REPSA的应用程序，用于确定孤儿转录调节因子的首选DNA结合位点，并确定其可能的生物学作用的第一步。
One of the first steps towards elucidating the biological function of a putative transcriptional regulator is to ascertain its preferred DNA-binding sequences. This may be rapidly and effectively achieved through the application of a combinatorial approach, one involving very large numbers of randomized oligonucleotides and reiterative selection and amplification steps to enrich for high-affinity nucleic acid-binding sequences. Previously, we had developed the novel combinatorial approach Restriction Endonuclease Protection, Selection and Amplification (REPSA), which relies not on the physical separation of ligand-nucleic acid complexes but instead selects on the basis of ligand-dependent inhibition of enzymatic template inactivation, specifically cleavage by type IIS restriction endonucleases. Thus, no prior knowledge of the ligand is required for REPSA, making it more amenable for discovery purposes. Here we describe using REPSA, massively parallel sequencing, and bioinformatics to identify the preferred DNA-binding sites for the transcriptional regulator SbtR, encoded by the TTHA0167 gene from the model extreme thermophile Thermus thermophilus HB8. From the resulting position weight matrix, we can identify multiple operons potentially regulated by SbtR and postulate a biological role for this protein in regulating extracellular transport processes. Our study provides a proof-of-concept for the application of REPSA for the identification of preferred DNA-binding sites for orphan transcriptional regulators and a first step towards determining their possible biological roles.