Kwong Stephen M, Firth Neville
School of Biological Sciences, University of Sydney, New South Wales 2006, Australia.
School of Biological Sciences, University of Sydney, New South Wales 2006, Australia.
Plasmid. 2015 Mar;78:17-25. doi: 10.1016/j.plasmid.2015.01.002. Epub 2015 Jan 26.
pSK41 is a prototypical 46-kb conjugative multiresistance plasmid of Staphylococcus aureus. The pSK41 replication initiation protein (Rep) is rate-limiting for plasmid replication, and its expression is negatively regulated by a small, non-coding antisense transcript, RNAI, that is complementary to the rep mRNA leader region. In this study, enzymatic probing was used to verify the predicted secondary structures of RNAI and its target RNA. We demonstrated that two stem-loop structures of RNAI, SLRNAI-II and SLRNAI-III, were important for inhibition. A putative U-turn motif detected in the loop of SLrep-I (5'-UUGG-3') was analysed for its significance to RNAI-mediated inhibition in vivo and Northern blotting suggested that rep mRNA was processed. Taken together, these observations support our previously proposed model but also raise new questions about the replication control mechanism.
pSK41是金黄色葡萄球菌一种典型的46千碱基的接合型多耐药性质粒。pSK41复制起始蛋白（Rep）对质粒复制起限速作用，其表达受到一种小的非编码反义转录本RNAI的负调控，RNAI与rep mRNA的前导区互补。在本研究中，酶促探测被用于验证RNAI及其靶RNA的预测二级结构。我们证明RNAI的两个茎环结构，即SLRNAI-II和SLRNAI-III，对抑制作用很重要。对在SLrep-I环（5'-UUGG-3'）中检测到的一个假定的U型转弯基序在体内对RNAI介导的抑制作用的意义进行了分析，Northern印迹法表明rep mRNA被加工了。综上所述，这些观察结果支持我们之前提出的模型，但也对复制控制机制提出了新的问题。
