转录调节蛋白,也被称为转录因子,作为分子开关调节基因表达的第一步,转录起始。环AMP受体蛋白(CRPs)和富马酸盐和硝酸盐还原调节剂(FNRs)组成了CRP/FNR超家族的转录因子,调节响应一系列刺激的基因表达。在目前的工作中,应用反向遗传学方法研究了模型生物嗜热菌HB8中4种CRP/FNR超家族转录因子之一的TTHA1359.限制性内切核酸酶保护,选择,和扩增(REPSA),然后是下一代测序技术和生物信息学基序发现,可以鉴定TTHA1359,5'-AWTGTRA(N)6TYACAWT-3'的DNA结合共识,TTHA1359以高亲和力结合。通过生物信息学将共识映射到嗜热T.thermophilusHB8基因组,在体外鉴定并验证了几个潜在的调节TTHA1359结合位点.这些发现有助于了解嗜热菌HB8中的TTHA1359调控活性,并证明了反向遗传方法在推定转录因子研究中的有效性。
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 Thermus thermophilus 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)6TYACAWT-3\', which TTHA1359 binds to with high affinity. By bioinformatically mapping the
consensus to the T. thermophilus 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 T. thermophilus HB8 and demonstrate the effectiveness of a reverse-genetic methodology in the study of putative transcription factors.