依赖PrfA转录调控的单核细胞增生李斯特菌毒力基因inlC启动子结构特点的初步研究

Some essential elements on the inlC promoter for PrfA-dependent regulation in Listeria monocytogenes

摘要:

为了研究单核细胞增生李斯特菌毒力基因启动子的结构特点与转录调控因子PrfA蛋白之间的关系，应用PCR定点突变和重组PCR技术缺失了该菌毒力基因inlC启动子上可能与PrfA蛋白结合以及诱发转录起始相关的碱基序列，构建了一系列突变启动子与lacZ报告基因融合表达质粒, 使lacZ基因的表达置于inlC突变启动子下，并分别电转化单核细胞增生李斯特菌野生株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。结果表明：位于inlC启动子转录起始点下游22bp 处的一段17bp的类似PrfA蛋白结合序列TTAACAGCGTTTGTTAA并没有增强和抑制PrfA转录调控活性的功能；甚至将其改造成“完美的” PrfA蛋白结合序列TTAACATTTGTTAA后，也不影响inlC依赖于PrfA的转录活性地表达；但是，如果缺失inlC启动子上原始的PrfA蛋白结合序列，则使inlC依赖于PrfA的转录活性完全丧失；另外，单核细胞增生李斯特菌毒力基因inlC和plcA 依赖于PrfA的转录活性的表达也与启动子上PrfA蛋白结合区(PrfA-box)距离-10区的碱基个数有关:最适为22或23bp，长于23bp或短于22bp的突变启动子的依赖PrfA的转录活性大大降低，甚至没有活性。说明除PrfA蛋白结合序列外，受PrfA调控的毒力基因启动子上还可能存在其它尚未阐明的结构和序列影响PrfA蛋白的结合以及启动转录表达。

Abstract:

To study some essential elements of a PrfA-dependent promoter of Listeria monocytogenes, a series of promoter mutants incorporated into upstream of a promoterless lacZ gene were constructed from a known listerial PrfA-dependent promoter, inlC promoter, by PCR-mediated site-directed mutagenesis and recombinant PCR technique and then electroporated into L. monocytogenes wild-type strain P14, prfA* mutant P14a and prfA deletion mutant A42.The corresponding transcription activities of altered promoters were measured by the β-galactosidase assay. The results showed that a PrfA-box-like sequence (“pseudo-PrfA-box”), TTAACAGCGTTTGTTAA, 22bp downstream of the transcriptional start site of PinlC had no ability to enhance or inhibit the PrfA-dependent transcription of inlC promoter, even it was modified to the “ideal PrfA-box” TTAACATTTGTTAA. However, there was almost no PrfA-dependent transcriptional activity from the mutants deletion of the inlC original PrfA-box. Moreover, altered spacing between 3′-end of the PrfA-box and 5′-end of the -10 box in the inlC promoter region affected transcription efficiency dramatically, which also happened in another promoter-dependent promoter, plcA promoter. Those above suggested that besides the “PrfA-box", additional unknown PrfA-dependent promoter structure(s) or sequence(s) might be required for the PrfA binding to the promoter and initiation of transcription. Furthermore, the distance between the PrfA-box and the -10 box should be fixed to 22 or 23bp for the PrfA-dependent transcription.