Compensatory mutations in the L30e kink-turn RNA-protein complex.
Compensatory mutations in the L30e kink-turn RNA-protein complex.
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DOI：
10.1016/j.bbagrm.2009.05.003
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发表时间：
2009-06
期刊：
影响因子：
--
通讯作者：
White SA
中科院分区：
文献类型：
--
作者：
Schweppe JJ;Jain C;White SA
色葡萄酿酒酵母核糖体蛋白L30 e是一种自身调节蛋白，其结合其自身的前mRNA和成熟mRNA以分别抑制剪接和翻译。L30 e RNA结合元件是形成扭结的茎-不对称环-茎。设计细菌遗传系统以测试蛋白变体抑制含有L30 e RNA结合元件的报告mRNA表达的能力。初步筛选显示，几种RNA核苷酸的变化对野生型蛋白质对报告基因的抑制具有可测量的影响。针对随机诱变的L30 e蛋白文库筛选减少阻遏的RNA突变体。这些筛选鉴定了L30 e的甘氨酸至丝氨酸突变，其特异性地恢复了含有取代螺旋加帽G的U的RNA变体的活性。类似地，发现天冬酰胺到丙氨酸的突变抑制L30 e RNA核苷酸延伸到蛋白质口袋中的位置处的取代。此外，一个有缺陷的RNA变体内的补偿性RNA突变被发现。这些抑制因子的鉴定提供了新的见解的架构的功能结合元件和它的识别的一个重要的RNA结合蛋白。
The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA binding element is a stem-asymmetric loop-stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.
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