Smith M C, Owen C E
Department of Biological and Molecular Science, University of Stirling, UK.
Mol Microbiol. 1991 Nov;5(11):2833-44. doi: 10.1111/j.1365-2958.1991.tb01992.x.
The sequence of the repressor locus, c, of the Streptomyces temperate phage, phi C31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in-frame N-terminally different, C-terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N-terminal sequencing, and used to raise antibody. The antibody cross-reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolor phi C31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super-infection immunity. Data described here and current knowledge of the nature of other 'dual start' genes suggest a model for the regulation of lysis versus lysogeny in phi C31.
此前已表明，温和型链霉菌噬菌体φC31的阻遏物基因座c的序列含有一个编码74 kDa蛋白的开放阅读框。对c基因转录和翻译产物的进一步分析显示出一种更为复杂的表达模式。发现从相应的转录本簇中表达出一窝三个N端不同、C端相同的蛋白，分子量分别为74 kDa、54 kDa和42 kDa。这些阻遏蛋白在大肠杆菌中产生，42 kDa的蛋白被纯化，通过N端测序进行验证，并用于制备抗体。该抗体在蛋白质免疫印迹中与在大肠杆菌、变铅青链霉菌中以及天蓝色链霉菌φC31溶源菌中表达的74 kDa、54 kDa和42 kDa蛋白发生交叉反应。通过S1作图和引物延伸对c基因转录的分析表明，编码阻遏蛋白的转录本簇在cts基因座受热处理后被诱导（Sinclair和Bibb，1989；本文）。相应地，所有三种阻遏蛋白都被诱导。除了位于74 kDa开放阅读框上游的启动子cp1外，c基因座还包含至少一个内部启动子cp2，它转录编码54 kDa和42 kDa蛋白的DNA。在含有缺失cp1和cp2的c基因的变铅青链霉菌的RNA制备物中观察到从cp3起始的转录本，但使用应含有该区域任何假定促进活性的DNA与xylE基因进行转录融合的基因融合体在变铅青链霉菌中显示儿茶酚2,3-双加氧酶的表达水平非常低。74 kDa的蛋白对于超感染免疫不是必需的。本文所述数据以及目前对其他“双起始”基因性质的了解提示了一个关于φC31中裂解与溶原调控的模型。
