Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1
Genetic analysis of the structure and function of transfer messenger RNA pseudoknot 1
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DOI：
10.1074/jbc.m600167200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Buskirk, AR
中科院分区：
文献类型：
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作者：
Tanner, DR;Dewey, JD;Buskirk, AR
tmRNA通过迫使核糖体放弃其mRNA模板并以tmRNA本身作为模板恢复翻译来拯救真细菌中停滞的核糖体。伪结1（pk1），紧接着上游的这个编码区的tmRNA，是一个结构元件，被认为是必不可少的tmRNA功能的基础上分析pk1突变体在体外。PK1在核糖体解码位点附近结合，并可与tmRNA配体进行碱基特异性接触。为了研究PK1在体内的结构和功能，我们开发了一种遗传选择，将大肠杆菌细胞的寿命与tmRNA活性联系起来。突变PK1在20%每个碱基和选择的tmRNA活性产生的序列，保留相同的假结倍。相比之下，从106个完全随机序列中选择活性突变体，鉴定了在功能上取代PK1的发夹结构。使用不相关序列合理设计具有增加的稳定性的发夹产生具有接近野生型活性的tmRNA突变体。我们的结论是，PK1在tmRNA功能的作用是纯粹的结构，它可以被替换为各种发夹结构。我们的研究结果表明，在功能性RNA的研究中，不应将旨在破坏给定结构的突变体的失活解释为该结构是RNA功能所必需的证据。这样的突变可能只会破坏一个整体折叠的稳定性，而这个整体折叠同样可以由一个完全不同的稳定结构形成。
tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 106 completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.