Characterization of conserved bases in 4.5S RNA of Escherichia coli by construction of new F' factors.
Characterization of conserved bases in 4.5S RNA of Escherichia coli by construction of new F' factors.
复制标题
通过构建新的 F 因子来表征大肠杆菌 4.5S RNA 中的保守碱基。
DOI：
10.1128/jb.00995-08
复制
发表时间：
2008
影响因子：
3.2
通讯作者：
Phillips,GregoryJ
中科院分区：
文献类型：
--
作者：
Peterson,JamesM;Phillips,GregoryJ
为了更清楚地了解 4.5S RNA（大肠杆菌必需基因的产物）保守碱基的功能，并解决其他研究报告的相互矛盾的结果，我们开发了一种新的遗传系统来表征突变体。通过改变与信号识别颗粒组装中的 Ffh 直接相互作用的 RNA 分子区域相对应的位置来生成多个 ffsalele。为了通过最少的操作来促进突变的表征，使用重组工程构建新的 F' 因子，以轻松地将每个等位基因移动到不同的遗传背景中，以便在单拷贝中表达。与以多个拷贝数表达 ffs 的质粒相结合，F' 因子可以准确评估不同 4.5S RNA 突变体在体内发挥作用的能力。与信号识别颗粒 (SRP) 的结构分析一致，4.5S RNA 中高度保守的碱基对于结合 Ffh 很重要。然而，尽管高度保守，但在所有测试条件下，只有一个碱基 (C62) 对于 RNA 功能是不可或缺的。为了量化 4.5S RNA 和 Ffh 之间的相互作用，开发了一种测定法来测量突变 4.5S RNA 分子与 Ffh 共纯化的能力。 Ffh 结合缺陷与 SRP 依赖性蛋白定位的丧失相关。还使用实时定量PCR来测量体内表达的野生型和突变型4.5S RNA的水平。这些结果澄清了先前研究的不一致之处，并提供了一种研究多个等位基因功能的便捷方法。
To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essentialffsgene ofEscherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterizeffsmutants. Multipleffsalleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of theffsmutations with minimal manipulation, recombineering was used to construct new F′ factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressedffsin multiple copy numbers, the F′ factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.