Kwong Stephen M, Skurray Ronald A, Firth Neville
School of Biological Sciences, Macleay Building A12, University of Sydney, Sydney, New South Wales 2006, Australia.
J Bacteriol. 2006 Jun;188(12):4404-12. doi: 10.1128/JB.00030-06.
Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (PrnaI) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when PrnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.
葡萄球菌多耐药质粒pSK41的复制受到反义转录本RNAI的负调控。携带rnaI启动子（PrnaI）突变的pSK41微型复制子的拷贝数显著增加，比野生型复制子的拷贝数高约40倍。使用rep控制区与cat报告基因之间的转录和翻译融合来评估RNAI突变对复制起始蛋白（Rep）表达的影响。结果表明，当PrnaI被破坏时，rep mRNA的量增加，并且其翻译变得去抑制。当反式提供RNAI时，这些效应被逆转，表明它在两个水平上负责显著的负调控，对rep翻译起始施加最大的抑制作用。诱变没有提供证据表明RNAI介导的转录衰减是观察到的与RNAI表达相关联的rep信息减少的基础。然而，RNA二级结构预测和支持性诱变数据表明了一种RNAI介导的rep翻译起始抑制的新机制，其中RNAI结合促进rep mRNA前导序列向另一种热力学稳定的茎环结构的空间转变，该结构隔离rep翻译起始区域，从而阻止翻译。
