Kolla V, Chakravorty M, Pandey B, Srinivasula S M, Mukherjee A, Litwack G
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, 221005, Varanasi, India.
Gene. 2000 Aug 22;254(1-2):209-17. doi: 10.1016/s0378-1119(00)00264-x.
MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.
MB78是鼠伤寒沙门氏菌的一种烈性噬菌体，具有许多有趣的特征，使其成为研究基因表达调控的合适生物体。已构建了该噬菌体基因组的详细物理图谱，并正在分子水平上进行广泛研究。在此，我们证明了噬菌体MB78的两种晚期蛋白源自同一基因，这可能是核糖体移码的结果。体外转录-翻译产生一种主要蛋白，其迁移率为28kDa，而使用pET表达载体的体内表达产生两种分子大小分别为28kDa和26kDa且表达量相同的蛋白。在阅读框中鉴定出一个假定的滑序列TTTAAAG和一个假结结构，这是经典核糖体移码所需的两个必需顺式元件。在滑序列处产生的突变导致单一的28kDa蛋白，并完全消除了26kDa蛋白的表达。因此，我们首次证明了核糖体移码发生在鼠伤寒沙门氏菌的噬菌体MB78中。
