Wu Cheng-Yeu, Fu Jen-Fen, Liu Shih-Tung
Department of Microbiology and Immunology, Chang-Gung University, Kwei-Shan, 333, Taiwan2.
Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei, 112, Taiwan1.
Microbiology (Reading). 2001 Oct;147(Pt 10):2757-2767. doi: 10.1099/00221287-147-10-2757.
A 2019 bp DNA fragment containing the replicon of pSW800 from Pantoea stewartii SW2 was cloned and characterized. This replicon contains two genes--repA and repB, which encode a 36.5 kDa replication initiation protein (RepA) and a peptide of 18 aa, respectively. These two genes overlap by 8 bases with repB situated upstream. The replicon also transcribes an antisense RNA (RNAI) that inhibits the expression of repA and repB. The ribosome-binding sequence (RBS) of repA is likely to be hidden in a stem-loop structure, inhibiting the translation of repA. Furthermore, translation of repB is likely to disrupt the stem-loop structure, which is one of the criteria allowing the translation of repA to begin. A mutagenesis study revealed that a sequence (5'-GCACGGG-3') located 111 nt upstream from repA is crucial; mutation of this sequence prevented the translation of repA. Additionally, this region and the stem-loop structure containing the RBS of repA may form an RNA pseudoknot. Results in this study demonstrate that a mechanism similar to that regulating plasmid replication in the IncB, IncIalpha and IncL/M groups also regulates pSW800 replication.
克隆并鉴定了一个包含来自斯氏泛菌SW2的pSW800复制子的2019 bp DNA片段。该复制子包含两个基因——repA和repB，它们分别编码一个36.5 kDa的复制起始蛋白（RepA）和一个18个氨基酸的肽段。这两个基因重叠8个碱基，repB位于上游。该复制子还转录一种反义RNA（RNAI），它抑制repA和repB的表达。repA的核糖体结合序列（RBS）可能隐藏在一个茎环结构中，从而抑制repA的翻译。此外，repB的翻译可能会破坏茎环结构，这是允许repA翻译开始的标准之一。诱变研究表明，位于repA上游111 nt处的一个序列（5'-GCACGGG-3'）至关重要；该序列的突变阻止了repA的翻译。此外，该区域和包含repA的RBS的茎环结构可能形成一个RNA假结。本研究结果表明，一种类似于调节IncB、IncIα和IncL/M组中质粒复制的机制也调节pSW800的复制。
