Hjalt T A, Wagner E G
Department of Microbiology, Uppsala University, Sweden.
Nucleic Acids Res. 1995 Feb 25;23(4):571-9. doi: 10.1093/nar/23.4.571.
Bulged-out nucleotides or internal loops are present in the stem-loop structures of several antisense RNAs. We have used the antisense/target RNA system (CopA/CopT) that controls the copy number of plasmid R1 to examine the possible biological function of bulged-out nucleotides. Two regions within the major stem-loop of the antisense RNA, CopA, carry bulged-out nucleotides. Base pairing in either one or both of these regions of the stem was restored by site-specific mutagenesis and in one case a new internal loop was introduced. The set of mutant and wild-type CopA variants was characterized structurally in vitro. The results reported here indicate a possible function of the bulges: their presence protects CopA RNA from being a substrate for the double-strand-specific enzyme RNase III. In vitro cleavage rates were drastically increased when either the lower or both bulges were absent. This is paralleled by a similar, but not identical, effect of the bulges on metabolic stability of the CopA RNAs in vivo. The degradation pathways of wild-type and mutant CopA in various strain backgrounds are discussed. In the accompanying paper, we address the significance of bulges in CopA for binding to the target RNA in vitro and for its inhibitory efficiency in vivo.
几个反义RNA的茎环结构中存在突出的核苷酸或内环。我们使用了控制质粒R1拷贝数的反义/靶RNA系统（CopA/CopT）来研究突出核苷酸可能的生物学功能。反义RNA CopA的主要茎环内的两个区域带有突出的核苷酸。通过位点特异性诱变恢复了茎的这两个区域中一个或两个区域的碱基配对，并且在一种情况下引入了新的内环。对一组突变型和野生型CopA变体进行了体外结构表征。此处报道的结果表明了突出部分的可能功能：它们的存在可保护CopA RNA不成为双链特异性酶RNase III的底物。当不存在下部突出部分或两个突出部分都不存在时，体外切割速率会急剧增加。这与突出部分对体内CopA RNA代谢稳定性的类似但不完全相同的影响并行。讨论了野生型和突变型CopA在各种菌株背景下的降解途径。在随附的论文中，我们探讨了CopA中突出部分对于体外与靶RNA结合及其体内抑制效率的意义。
