单核细胞增生李斯特菌毒力基因aroA启动子上阻碍PrfA转录调控元件的研究  

Study on inhibitory elements of a putative PrfA-dependent promoter ParoA2 in Listeria monocytogenes

作　　者：罗勤[1] 周青春[1] 

机构地区：[1]华中师范大学生命科学学院,遗传调控与整合生物学湖北省重点实验室,武汉430079

出　　处：《中华微生物学和免疫学杂志》2008年第6期521-527,共7页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金项目（30500025）;教育部留学回国人员科研启动基金;教育部211重点学科项目资助

摘　　要：目的研究食源性致病菌单核细胞增生李斯特菌（Listeria monocytogenes，简称LM）毒力基因启动子的结构特点及其与转录调控因子PrfA（positive regulatory factorA）蛋白之间的关系。方法选取picA和aroA两个毒力基冈启动子作为研究对象，picA基因启动子（PplcA）上含有一个高度保守的PrfA蛋白结合序列TFAACAAATGTFAA（PrfA-box）和-10区（TAAGAT），其转录表达受PrfA强调控；aroA基因不受PrfA调控，所利用的启动子ParoA1为非依赖于PrfA的启动子，但是在紧邻ParoA1的下游区域却含有一个与PplcA相似的PrfA—box（TTAAAACATGTTAA）和一个-10区（TTTAAT），该区域疑似为依赖于PrfA的启动子，被命名为ParoA2。应用PCR定点突变和SOEing PCR（重叠区扩增基因拼接法）技术互换了Pplca和ParoA2上可能影响PrfA蛋白结合以及诱发转录起始相关的碱基序列，构建了一系列PplcA—ParoA2杂合突变启动子，并插入到无启动子的lacZ报告基因上游，使lacZ基因的表达置于突变启动子下。获得的启动子融合表达质粒分别电转化入LM野生株P14、PrfA蛋白高表达突变株P14a和prfA基因等位缺失突变株A42中，检测相应的β-半乳糖苷酶活性以确定杂合突变启动子是否具有依赖PrfA的转录活性及其水平高低。结果当启动子上影响PrfA转录调控的两个核心元件PrfA—box与-10区的距离保持在最适的22或23个碱基时，交换印M和ParoA2上的两个相应核心元件的碱基序列并不改变启动子的转录活性。但是，当PplcA上的两个核心元件之间的碱基以及-10区下游的序列被相应ParoA2上的序列所替代后，PplcA依赖于PrfA的转录活性完全丧失，反之，ParoA2上的这两段序列如果被PplcA的序列所替换，ParoA2则表现出依赖于PrfA的转录活性。结论ParoA2的-10区及其下游的序列可能形成发夹结构，阻碍RNA聚合酶-PrfA蛋白复合物结合到解旋的单链模板DNA上生成具有转录起�Objective To investigate the relationship between PrfA-dependent promoters and PrfA regulation. Methods LacZ reporter gene fusions used to investigate the inhibitory elements for PrfA-dependent transcription were carried on two promoters of Listeria monocytogenes ： a PrfA-dependent promoter of the phospholipase gene picA （ PplcA ） and a putative promoter of the aroA gene （ ParoA2 ） which contains a similar PrfA-binding site （PrfA-box） and a similar -10 box as PplcA but does not function as PrfA-dependent promoter. A series of hybrid plcA-aroA promoters by exchanging corresponding sequence elements of these two ＂promoters＂ were constructed and incorporated into upstream of a promoterless lacZ gene. The variant promoter-lacZ transcriptional fusions were then electroporated into L. monoeytogenes wild-type strain P14, prfA mutant P14a and prfA deletion mutant A42, respectively. The expression level of PrfA is the highest in the P14a and the lowest in A42. The corresponding transcription activities of hybrid promoters were measured by the β-galactosidase assay. Results The two critical elements of PrfA-dependent promoters, the PrfA-box and the -10 box, can be functionally exchanged as long as the distance in between is maintained 22 or 23 bp. However, the interspace sequence and the sequence downstream of the -10 box of ParoA2 were strongly inhibitory for PrfA-dependent transcription. Conclusion Downstream sequence together -10 box of ParoA2 might fold into a hairpin structure when present in a single stranded DNA and possibly block the formation of the transcriptional initiation open complex, hence, inhibit the PrfA-dependent transcription from ParoA2.

关 键 词：单核细胞增生李斯特菌 PrfA 依赖于PrfA的启动子 转录调控 

分 类 号：R516[医药卫生—内科学]