Regulation of the EcoRI restriction-modification system:: Identification of ecoRIM gene promoters and their upstream negative regulators in the ecoRIR gene
Regulation of the EcoRI restriction-modification system:: Identification of ecoRIM gene promoters and their upstream negative regulators in the ecoRIR gene
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DOI：
10.1016/j.gene.2007.06.006
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发表时间：
2007-10-01
期刊：
影响因子：
3.5
通讯作者：
Kobayashi, Ichizo
中科院分区：
文献类型：
--
作者：
Liu, Yaoping;Ichige, Asao;Kobayashi, Ichizo
II型限制-修饰（R-M）系统由连接的限制性内切酶和修饰甲基转移酶基因组成，并作为水平基因转移的屏障，即使它们本身是移动的。他们的产品杀死已经失去R-M基因的宿主细菌细胞，这一过程有助于维持活细胞群中R-M系统的频率。它们在细菌宿主中的建立和维持预期涉及对其基因表达的精细调控。在本研究中，我们分析了该修饰基因的转录及其在EcoRIR-M系统中的调控。北方印迹显示，下游的ecoRIM基因被转录为单顺反子mRNA，并作为一个更大的双顺反子mRNA的一部分与上游的ecoRIR基因。引物延伸、RNA酶保护和使用lacZ基因融合的突变分析鉴定了在ecoRIR基因内用于ecoRIM基因转录的两个重叠启动子。进一步的突变分析显示，在ecoRIR基因内的两个上游AT丰富的元件，“AATAAA”和“ATTATAAATATA”，作为这些启动子的负调控因子发挥作用。这两个元素的同时取代导致β-半乳糖苷酶活性增加四倍，转录水平增加五倍，如通过RNA酶保护测定所测量的。对ecoRIM转录物的RNA测量表明，这些元件通过干扰ecoRIM启动子的转录起始来降低ecoRIM表达。这些ecoRIM启动子和他们的负调控EcoRIR-M系统中的可能作用进行了讨论。(c)2007 Elsevier B. V.保留所有权利。
Type II restriction-modification (R-M) systems are composed of linked restriction endonuclease and modification methyltransferase genes and serve as barriers to horizontal gene transfer even though they are mobile in themselves. Their products kill host bacterial cells that have lost the R-M genes, a process that helps to maintain the frequency of the R-M systems in the viable cell population. Their establishment and maintenance in a bacterial host are expected to involve fine regulation of their gene expression. In the present study, we analyzed transcription of the modification gene and its regulation within the EcoRIR-M system. Northern blotting revealed that the downstream ecoRIM gene is transcribed as a monocistronic mRNA and as part of a larger bicistronic mRNA together with the upstream ecoRIR gene. Primer extension, RNase protection, and mutational analysis using lacZ gene fusions identified two overlapping promoters for ecoRIM gene transcription within the ecoRIR gene. Further mutational analysis revealed that two upstream AT-rich elements within the ecoRIR gene, "AATAAA" and "ATTATAAATATA," function as negative regulators of these promoters. Simultaneous substitution of these two elements resulted in a fourfold increase in beta-galactosidase activity and a five-fold increase in transcript levels as measured by RNase protection assay. RNA measurements of the ecoRIM transcript suggested that these elements decreased ecoRIM expression by interfering with transcription initiation of the ecoRIM promoters. Possible roles for these ecoRIM promoters and their negative regulators in the EcoRIR-M system are discussed. (c) 2007 Elsevier B.V. All rights reserved.