The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA
The regulatory TnaC nascent peptide preferentially inhibits release factor 2-mediated hydrolysis of peptidyl-tRNA
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DOI：
10.1074/jbc.ra119.011313
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发表时间：
2019-12-13
影响因子：
4.8
通讯作者：
Cruz-Vera, Luis Rogelio
中科院分区：
文献类型：
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作者：
Emmanuel, Jerusha Salome;Sengupta, Arnab;Cruz-Vera, Luis Rogelio
来自大肠杆菌tna操纵子的tnaC调控基因在抑制其自身翻译终止时，通过依赖于捕获核糖体的积累的衰减机制来控制其自身操纵子的转录。这种依赖于游离L-Trp的翻译终止抑制机制尚不清楚。在这里，我们分析了l -色氨酸对两种已知的大肠杆菌翻译终止因子RF1和RF2功能的抑制作用。利用一系列报告基因，我们发现tnaC基因表达的体内L-Trp敏感性受其停止密码子的同一性影响，UGA停止密码子比UAG停止密码子产生更高的tnaA-lacZ基因结构的表达效率。体外tnc -peptidyl- trna积累和脚印实验证实，在L-Trp存在下，UGA终止密码子比UAG终止密码子产生更高的tnc -peptidyl- trna和捕获核糖体的积累。rf介导的水解实验证实，L-Trp比RF1更能阻断RF2的功能。突变分析表明，RF2-GGQ功能基序周围246和256个残基位置的氨基酸替换降低了依赖L-Trp的tnaC(UGA) tnaA-lacZ结构的表达，以及L-Trp抑制rf2介导的tnaC -肽基- trna切割的能力。总之，我们的研究结果表明，在tnaC基因的翻译终止过程中，L-Trp优先阻断RF2活性。这种抑制取决于RF2-GGQ功能基序周围氨基酸残基的身份。
The tnaC regulatory gene from the tna operon of Escherichia coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. This free L-Trp dependent mechanism of inhibition of translation termination remains unclear. Here, we analyzed the inhibitory effects of L-Trp on the function of two known E. coli translation termination factors, RF1 and RF2. Using a series of reporter genes, we found that the in vivo L-Trp sensitivity of tnaC gene expression is influenced by the identity of its stop codon, with the UGA stop codon producing higher expression efficiency of the tnaA-lacZ gene construct than the UAG stop codon. in vitro TnaC-peptidyl-tRNA accumulation and toeprinting assays confirmed that in the presence of L-Trp, the UGA stop codon generates higher accumulation of both TnaC-peptidyl-tRNA and arrested ribosomes than does the UAG stop codon. RF-mediated hydrolysis assays corroborated that L-Trp blocks RF2 function more than that of RF1. Mutational analyses disclosed that amino acids substitutions at the 246 and 256 residue positions surrounding the RF2-GGQ functional motif reduce L-Trp dependent expression of the tnaC(UGA) tnaA-lacZ construct and the ability of L-Trp to inhibit RF2-mediated cleavage of the TnaC-peptidyl-tRNA. Altogether, our results indicate that L-Trp preferentially blocks RF2 activity during translation termination of the tnaC gene. This inhibition depends on the identities of amino acid residues surrounding the RF2-GGQ functional motif.