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首页> 外文期刊>Journal of bacteriology >Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.
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Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

机译：质粒pGKV21在大肠杆菌中的ssi信号的功能特征。

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A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.
机译：在包含pWV01复制子的乳酸乳球菌质粒pGKV21上检测到单链起始（ssi）信号，原因是它能够补充大肠杆菌中缺乏互补链来源的M13噬菌体衍生物（M13 delta lac182）的不良生长。该ssi信号位于229个核苷酸（nt）的DdeI-DraI片段上，并且位于推定的+起源的切口位点上游的109nt内。 SSI活动相对于复制方向是特定于方向的。我们构建了一个缺失ssi信号的质粒，然后检查了ssi信号对大肠杆菌中单链复制中间体向双链质粒DNA转化的影响。缺少ssi信号的质粒比野生型质粒积累的质粒单链DNA多得多。此外，该区域的缺失引起质粒拷贝数或质粒维持性的极大降低。这些结果表明，在大肠杆菌中，此ssi信号将其滞后链合成指导为质粒pGKV21的负来源。体外引物RNA合成表明，大肠杆菌RNA聚合酶可直接识别229-nt ssi信号，并根据大肠杆菌单链DNA结合（SSB）蛋白的存在来合成引物RNA。该区域包含两个茎环结构，茎环I和茎环II。茎环I部分的缺失会导致大肠杆菌RNA聚合酶失去引物活性，这表明茎环I部分对于由SSB包被的单链DNA模板上的大肠杆菌RNA聚合酶引物至关重要。

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