Department of Chemistry and Biochemistry, Kennesaw State University, Kennesaw, GA 30144, USA.
Int J Mol Sci. 2021 Sep 17;22(18):10042. doi: 10.3390/ijms221810042.
Transcription regulatory proteins, also known as transcription factors, function as molecular switches modulating the first step in gene expression, transcription initiation. Cyclic-AMP receptor proteins (CRPs) and fumarate and nitrate reduction regulators (FNRs) compose the CRP/FNR superfamily of transcription factors, regulating gene expression in response to a spectrum of stimuli. In the present work, a reverse-genetic methodology was applied to the study of TTHA1359, one of four CRP/FNR superfamily transcription factors in the model organism HB8. Restriction Endonuclease Protection, Selection, and Amplification (REPSA) followed by next-generation sequencing techniques and bioinformatic motif discovery allowed identification of a DNA-binding consensus for TTHA1359, 5'-AWTGTRA(N)TYACAWT-3', which TTHA1359 binds to with high affinity. By bioinformatically mapping the consensus to the HB8 genome, several potential regulatory TTHA1359-binding sites were identified and validated in vitro. The findings contribute to the knowledge of TTHA1359 regulatory activity within HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.
转录调控蛋白，也被称为转录因子，作为分子开关调节基因表达的第一步，即转录起始。环磷酸腺苷受体蛋白（CRPs）和延胡索酸和硝酸盐还原调节蛋白（FNRs）构成 CRP/FNR 转录因子超家族，调节基因表达以响应一系列刺激。在本工作中，应用反向遗传学方法研究了模式生物 HB8 中四个 CRP/FNR 转录因子之一的 TTHA1359。限制内切酶保护、选择和扩增（REPSA），随后是下一代测序技术和生物信息学基序发现，鉴定了 TTHA1359 的 DNA 结合保守序列为 5'-AWTGTRA(N)TYACAWT-3'，TTHA1359 与之具有高亲和力结合。通过将保守序列在生物信息学上映射到 HB8 基因组上，鉴定并验证了几个潜在的 TTHA1359 结合的调控位点。这些发现有助于了解 TTHA1359 在 HB8 中的调控活性，并证明了反向遗传学方法在研究潜在转录因子中的有效性。
