Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.
Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.
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参与尾部完成的噬菌体 P2 基因 R 和 S 的分子克隆和表征。
DOI：
10.1006/viro.1994.1199
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发表时间：
1994
期刊：
影响因子：
3.7
通讯作者：
Christie,GE
中科院分区：
文献类型：
--
作者：
Linderoth,NA;Julien,B;Flick,KE;Calendar,R;Christie,GE
测定了温带噬菌体P2的两个已知尾部基因rands的序列和位于SandV之间的另一个开放阅读框(orf-30)的序列。对RandS、Ram3、Ram42、Ram23、Sam75和Sam89中的琥珀突变进行了测序，发现它们位于相应的开放阅读框内。我们构建了R和S的高产表达载体，并通过全细胞裂解产物的十二烷基硫酸钠-PAGE和考马斯亮蓝染色对其进行了鉴定。预测的蛋白质R和S的相对分子质量分别为17,400和17,300，尽管这两个多肽在凝胶电泳过程中的迁移速度比从序列数据预期的要慢。σ-30占据了与RSandV相反的一条链，前面有几个潜在的弱启动子，其中一些与V启动子重叠。在LacZ基因上游含有β-30启动子区的构建体在体内产生低水平的LacZ-半乳糖苷酶活性。噬菌体P4转录激活蛋白δ不能刺激Theorf-30启动子的表达，该蛋白作用于所有已知的P2和P4晚期启动子。插入突变表明orf-30不是P2在大肠杆菌中生长所必需的基因。没有一个基因或蛋白质序列与核酸和蛋白质数据库中的序列有广泛的同源性。然而，R蛋白包含一个与噬菌体T4尾蛋白GP15中的一个同源的小区域，这是T4尾巴结合头部所必需的。我们认为R和S是稳定头部连接所必需的尾部完成蛋白。
The sequences of two previously known tail genes,RandS, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located betweenSandV, were determined. Amber mutations mapping withinRandS,Ram3,Ram42,Ram23,Sam75, andSam89were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S wereMr17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data.orf-30occupies the strand opposite fromRSandVand is preceded by several weak potential σ70-RNA polymerase promoters, some of which overlap with theVpromoter. A construct that had the putativeorf-30promoter region upstream of thelacZgene produced low levels of β-galactosidase activityin vivo. Expression from theorf-30promoter was not stimulated by the phage P4 transcriptional activator protein, δ, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth inEscherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.