结核分枝杆菌转录调控蛋白Rv0324调控rv3597c的初步研究

Preliminary research of Mycobacterium tuberculosis transcriptional protein Rv0324 reveals that it regulates rv3597c

结核分枝杆菌(MTB)利用其转录调控系统快速适应环境变化,而其Ars R家族的转录调控蛋白Rv0324则在转录调控系统中发挥着重要的作用。为研究Rv0324的调控机制,本研究以卡介苗(BCG)(Tokyo-172)基因组为模板,PCR扩增得到rv0324基因,构建p ET22b-rv0324重组表达载体,转化至E.coli BL21后经诱导表达获得蛋白Rv0324;同时以BCG(Tokyo-172)基因组为模板扩增获得rv3597c启动子(p/o rv3597c)。通过体外凝胶迁移实验(EMSA)和体内染色质免疫沉淀(Ch IP)实验对Rv0324与p/o rv3597c的相互作用进行检测,结果显示Rv0324可与p/o rv3597c结合,且Rv0324结合于p/o rv3597c的大沟;通过EMSA对影响Rv0324和p/o rv3597c结合的小分子进行筛选,结果显示二者的结合均能够被Trp、Fe^(3+)、Cu^(2+)、V_(B1)、V_(C)和贝达喹啉小分子抑制。在利用tr Rosetta软件对Rv0324蛋白结构分析的基础上,通过Rv0324和小分子的对接实验进一步分析它们间可能的互作模式,结果显示主要是位于Rv0324环区结构域的Asp203和Asp204氨基酸残基参与了与小分子的互作。综上所述,本研究首次证实MTB Rv0324通过直接调控rv3597c的表达,参与调控MTB的氧耗和代谢水平的变化,同时发现了影响Rv0324和rv3597c互作的小分子及其作用模式,为临床治疗的药物筛选提供了一定的参考依据。

Mycobacterium tuberculosis (MTB),the causative agent of tuberculosis,can adapt to changes in host is that transcription factors play an important role.Ars R family member Rv0324 is considered as a significant transcriptional regulator in MTB.However,its exactly function is not clear so far.To investigate the transcriptional mechanism of Rv0324,we constructed the recombinant vector p ET22b-rv0324 by PCR amplification of rv0324 from BCG (Tokyo-172) genome and then obtained protein Rv0324.And we also obtained promotor region of rv3597c (p/o rv3597c) by PCR amplification.Based on obtainment of recombinant protein and promotor region of rv3597c,we found that Rv0324 bound to p/o rv3597c by electrophoretic mobility shift assay (EMSA) in vitro and chromatin immunoprecipitation assay (Ch IP) in vivo.And DNA major/minor groove tests showed that Rv0324 bound to p/o rv3597c major groove.We also found that Trp,Fe^(3+),Cu^(2+),V_(B1)and V_(C)inhibited the interaction between Rv0324 and p/o rv3597c by EMSA screening tests.Furthermore,based on the Rv0324 homology model,molecular docking predict that Asp203 and Asp204 located in loop region are mainly involved in interaction between Rv0324 and cofactors (small molecules).In conclusion,Rv0324 directly regulates rv3597c and it is supposed that Rv0324 may be involved in the regulation of oxygen consumption and metabolism,which provides a theoretical foundation for drug screen of clinical treatment.