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首页> 外文期刊>Journal of Molecular Biology >Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA.
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Functional and structural analysis of a pseudoknot upstream of the tag-encoded sequence in E. coli tmRNA.

机译：大肠杆菌tmRNA中标签编码序列上游的假结的功能和结构分析。

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摘要

Escherichia coli tmRNA (transfer-messenger RNA) facilitates a trans-translation reaction in which a stalled ribosome on a terminatorless mRNA switches to an internal coding sequence in tmRNA, resulting in the addition of an 11 amino acid residue tag to the truncated protein that is a signal for degradation and in recycling of the stalled ribosome. A tmRNA secondary structure model with a partial tRNA-like structure and several pseudoknots was recently proposed. This report describes an extensive mutational analysis of one predicted pseudoknot (PK1) located upstream of the E. coli tmRNA tag-encoded sequence. Both the extent of aminoacylation and the alanine incorporation into the tag sequence, reflecting the two functions of tmRNA, were measured in vitro for all the engineered RNA variants. To characterize structure-function relationships for the tmRNA mutants, their solution conformations were investigated by using structural probes and by measuring the temperature dependence of their UV absorbance. This analysis strongly supports the presence of a pseudoknot in E. coli tmRNA, and its involvement in trans-translation. Mutations disrupting the first stem of the pseudoknot inactivate function and promote stable alternative conformations. Mutations of the second stem of the pseudoknot also effect both functions. The nucleotide stretch between the two stems (loop 2) is required for efficient trans-translation, and nucleotides at positions 61 and 62 must be guanine residues. The probing data suggest the presence of magnesium ion(s) interacting with loop 2. The loops crossing the minor and major grooves can be mutated without significant effects on tmRNA function. Nucleotide insertion or deletion between the pseudoknot and the coding sequence do not change the mRNA frame of the tag-peptide sequence, suggesting that the pseudoknot structure is not a determinant for the resumption of translation. Copyright 1999 Academic Press.
机译：大肠杆菌tmRNA（传递信使RNA）促进了反翻译反应，其中无终止子mRNA上停滞的核糖体切换到tmRNA的内部编码序列，从而在截短的蛋白质上增加了11个氨基酸残基标签。降解和停止的核糖体回收的信号。最近提出了具有部分tRNA样结构和几个假结的tmRNA二级结构模型。该报告描述了位于大肠杆菌tmRNA标签编码序列上游的一个预测的假结（PK1）的广泛突变分析。对于所有工程化的RNA变体，都在体外测量了反映酰化tmRNA的两种功能的氨基酰化程度和丙氨酸掺入标签序列中的程度。为了表征tmRNA突变体的结构-功能关系，通过使用结构探针并测量其UV吸光度的温度依赖性来研究其溶液构象。该分析强烈支持在大肠杆菌tmRNA中存在假结及其参与反式翻译。突变破坏假结的第一个茎使功能失活并促进稳定的替代构象。假结第二茎的突变也影响这两个功能。为了有效地进行翻译，需要两个茎之间的核苷酸延伸（环2），并且61和62位的核苷酸必须是鸟嘌呤残基。探测数据表明存在与环2相互作用的镁离子。跨小槽和大槽的环可以突变，而对tmRNA功能无明显影响。假结和编码序列之间的核苷酸插入或缺失不会改变标签肽序列的mRNA框架，这表明假结结构不是恢复翻译的决定因素。版权所有1999，学术出版社。

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