Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973
Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973
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DOI：
10.3390/ijms20133336
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发表时间：
2019-07-01
影响因子：
5.6
通讯作者：
Van Dyke, Michael W.
中科院分区：
文献类型：
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作者：
Cox, James Shell;Moncja, Kristi;Van Dyke, Michael W.
基因组测序的进步使得能够鉴定出编码假定的转录调节蛋白的多种基因。通常，人们缺乏对这些蛋白质、它们所调节的基因或调节子所发挥的生物学作用的更全面的了解。传统上，这是通过涉及推定转录因子基因操作和观察生物体转录组变化的遗传方法来实现的。然而，这种方法并不总是可行，或者可能会产生误导性的结果。在这里，我们描述了一种以生物化学为中心的方法，包括使用限制性核酸内切酶保护、选择和扩增（REPSA）选择方法、大规模并行测序和生物信息学分析来鉴定嗜热栖热菌 HB8 转录抑制子 TTHA0973 的首选 DNA 结合序列。我们鉴定了 5'-AACnAACGTTnGTT-3' 的共有 TTHA0973 识别序列，其表现出纳摩尔级的结合亲和力。该序列被定位到嗜热菌 HB8 基因组内的多个位点，其中的一个子集对应于调节参与苯乙酸降解的基因的启动子区域。这些研究进一步证明了以生物化学为中心的方法可用于轻松识别各种生物体中孤儿转录因子的潜在生物学功能。
Advances in genomic sequencing have allowed the identification of a multitude of genes encoding putative transcriptional regulatory proteins. Lacking, often, is a fuller understanding of the biological roles played by these proteins, the genes they regulate or regulon. Conventionally this is achieved through a genetic approach involving putative transcription factor gene manipulation and observations of changes in an organism's transcriptome. However, such an approach is not always feasible or can yield misleading findings. Here, we describe a biochemistry-centric approach, involving identification of preferred DNA-binding sequences for the Thermus thermophilus HB8 transcriptional repressor TTHA0973 using the selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), massively parallel sequencing, and bioinformatic analyses. We identified a consensus TTHA0973 recognition sequence of 5 '-AACnAACGTTnGTT-3 ' that exhibited nanomolar binding affinity. This sequence was mapped to several sites within the T. thermophilus HB8 genome, a subset of which corresponded to promoter regions regulating genes involved in phenylacetic acid degradation. These studies further demonstrate the utility of a biochemistry-centric approach for the facile identification of potential biological functions for orphan transcription factors in a variety of organisms.