Antisense RNA-mediated transcriptional attenuation:: an in vitro study of plasmid pT181
Antisense RNA-mediated transcriptional attenuation:: an in vitro study of plasmid pT181
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DOI：
10.1046/j.1365-2958.2000.01813.x
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发表时间：
2000-03-01
影响因子：
3.6
通讯作者：
Wagner, EGH
中科院分区：
文献类型：
--
作者：
Brantl, S;Wagner, EGH
反义RNA通过几种不同的机制调控质粒复制。其中一个机制，转录抑制，首先被描述为葡萄球菌质粒pT181，后来被描述为链球菌质粒pIP501和Pam beta 1。在此之前，我们对pIP501系统进行了详细的体外和体内分析。在这里，我们提出了一种对pT181反义系统的体外分析。测定了不同长度的反义和正义RNA物种的二级结构。测定了正义/反义RNA对的结合速率常数，并确定了形成复合体所需的功能片段。单轮转录实验用于体外转录衰减分析。PT181和pIP501之间的比较显示了一些差异；虽然pIP501反义RNA的截断衍生物足以稳定地形成复合体，但pT181-RNAi的两种茎环结构都是必需的。与pIP501的正义RNA显示出内在的终止倾向(在没有反义RNA的情况下为30-50%)相反，pT181的正义RNA需要反义RNA来诱导终止。PT181正义-反义RNA复合物的形成速率常数与抑制速率常数相似，这与pIP501形成的抑制速率常数形成鲜明对比，后者的抑制速度至少是稳定结合的10倍。
Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAM beta 1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the antisense system of plasmid pT181. The secondary structures of antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 antisense RNA was sufficient for stable complex formation, both stem-loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30-50% in the absence of antisense RNA), the sense RNA of pT181 required antisense RNA for induced termination. Rate constants of formation of pT181 sense-antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.