Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription
Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription
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DOI：
10.3791/59687
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发表时间：
2019-07-01
影响因子：
1.2
通讯作者：
Fan, Huizhou
中科院分区：
文献类型：
--
作者：
Desai, Malhar;Di, Rong;Fan, Huizhou
转录因子（TF）是一种通过与RNA聚合酶、另一种TF和/或模板DNA相互作用来调节基因表达的蛋白质。GrgA是一种新的转录激活因子，专门在专性细胞内细菌病原体衣原体中发现。使用亲和珠进行的蛋白下拉试验显示，GrgA结合了两个sigma因子，即sigma（66）和sigma(28)，它们识别不同的启动子组，这些启动子组的产物在发育阶段是不同的。我们使用BLI来确认和进一步表征相互作用。与下拉法相比，BLI有几个优势：1)它可以实时显示结合伙伴之间的关联和解离，2)它可以生成定量动力学参数，3)它可以检测下拉法通常无法检测到的结合。这些特征使我们能够推断GrgA在衣原体基因表达调控中的生理作用，以及可能的详细相互作用机制。我们设想这种相对便宜的技术对于研究转录和其他生物过程非常有用。
A transcription factor (TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two sigma factors, namely sigma(66) and sigma(28), which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.