Liu Yaoping, Kobayashi Ichizo
Department of Medical Genome Sciences, Graduate Schol of Frontier Science, University of Tokyo, Tokyo 108-8639, Japan.
J Bacteriol. 2007 Oct;189(19):6928-35. doi: 10.1128/JB.00127-07. Epub 2007 Jul 6.
Type II restriction-modification systems are expected to possess mechanisms for tight regulation of their expression to suppress the potential of lethal attack on their host bacteria when they establish and maintain themselves within them. Although the EcoRI restriction enzyme has been well characterized, regulation of its expression is still poorly understood. In this study, mutational analysis with lacZ gene fusion and primer extension assay identified a promoter for the transcription of the ecoRIR gene. Further analyses revealed that an intragenic region containing two overlapping reverse promoter-like elements acted as a negative regulator for ecoRIR gene expression. The activity of these putative reverse promoters was verified by transcriptional gene fusion, primer extension and in vitro transcription. Mutations in these reverse promoters resulted in increased gene expression in both translational and transcriptional gene fusions. An RNase protection assay revealed that the transcript level of the wild type relative to that of the reverse promoter mutant at the downstream regions was much lower than the level at the upstream regions. This suggests that these reverse promoter-like elements affect their downstream transcript level. The possible mechanisms of this kind of negative regulation, in addition to their possible biological roles, are discussed.
II型限制-修饰系统有望具备严格调控其表达的机制，以便在其于宿主细菌内建立并维持自身时，抑制对宿主细菌进行致命攻击的可能性。尽管EcoRI限制酶已得到充分表征，但其表达调控仍知之甚少。在本研究中，通过lacZ基因融合的突变分析和引物延伸试验鉴定出了ecoRIR基因转录的一个启动子。进一步分析表明，一个包含两个重叠的类反向启动子元件的基因内区域作为ecoRIR基因表达的负调控因子发挥作用。这些假定的反向启动子的活性通过转录基因融合、引物延伸和体外转录得以验证。这些反向启动子中的突变导致翻译和转录基因融合中的基因表达均增加。核糖核酸酶保护试验表明，野生型相对于反向启动子突变体在下游区域的转录本水平远低于上游区域的水平。这表明这些类反向启动子元件会影响其下游转录本水平。本文讨论了这种负调控的可能机制及其可能的生物学作用。
