SHAPE analysis of the htrA RNA thermometer from Salmonella enterica.
SHAPE analysis of the htrA RNA thermometer from Salmonella enterica.
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DOI：
10.1261/rna.062299.117
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发表时间：
2017-10
期刊：
影响因子：
--
通讯作者：
Mitton-Fry RM
中科院分区：
文献类型：
--
作者：
Choi EK;Ulanowicz KA;Nguyen YAH;Frandsen JK;Mitton-Fry RM
RNA温度计调节与致病菌（如耶尔森氏菌、奈瑟氏菌和沙门氏菌）毒力相关的一些基因的表达。它们通常通过改变核糖体结合位点的可及性的温度依赖性构象变化来起作用。肠道沙门氏菌htrA mRNA的5′-非翻译区（UTR）含有一个非常短的RNA温度计。我们已经系统地表征了这种温度计的结构和动力学，在单核苷酸分辨率使用SHAPE（选择性2′-羟基酰化引物延伸分析）测定。我们的研究结果证实，htrA温度计在低温下采用预测的发夹构象，在生理温度范围内发生构象变化。单个核苷酸的详细SHAPE熔解曲线表明，温度计以合作的方式展开，茎的上部和下部的核苷酸在共同的转变温度下获得灵活性。有趣的是，对延伸的htrA 5′ UTR序列的分析不仅揭示了RNA温度计的存在，而且还揭示了额外的稳定上游结构。我们生成并分析了htrA温度计的点突变体，揭示了调节其稳定性的元素，使发夹在温血宿主感染期间经历的略微升高的温度下融化。这项工作揭示了htrA和相关温度计的结构-功能关系，它也说明了SHAPE测定的实用性，用于RNA温度计系统的详细研究。
RNA thermometers regulate expression of some genes involved in virulence of pathogenic bacteria such as Yersinia, Neisseria, and Salmonella. They often function through temperature-dependent conformational changes that alter accessibility of the ribosome-binding site. The 5′-untranslated region (UTR) of the htrA mRNA from Salmonella enterica contains a very short RNA thermometer. We have systematically characterized the structure and dynamics of this thermometer at single-nucleotide resolution using SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) assays. Our results confirm that the htrA thermometer adopts the predicted hairpin conformation at low temperatures, with conformational change occurring over a physiological temperature regime. Detailed SHAPE melting curves for individual nucleotides suggest that the thermometer unfolds in a cooperative fashion, with nucleotides from both upper and lower portions of the stem gaining flexibility at a common transition temperature. Intriguingly, analysis of an extended htrA 5′ UTR sequence revealed not only the presence of the RNA thermometer, but also an additional, stable upstream structure. We generated and analyzed point mutants of the htrA thermometer, revealing elements that modulate its stability, allowing the hairpin to melt under the slightly elevated temperatures experienced during the infection of a warm-blooded host. This work sheds light on structure–function relationships in htrA and related thermometers, and it also illustrates the utility of SHAPE assays for detailed study of RNA thermometer systems.
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影响因子：
3.2
作者：
ALATOSSAVA, T;JUTTE, H;KELLENBERGER, E
通讯作者：
KELLENBERGER, E
影响因子：
4.1
作者：
Klinkert, Birgit;Cimdins, Annika;Narberhaus, Franz
通讯作者：
Narberhaus, Franz
影响因子：
4.5
作者：
Karabiber, Fethullah;McGinnis, Jennifer L.;Weeks, Kevin M.
通讯作者：
Weeks, Kevin M.
影响因子：
2.8
作者：
Lewis, Claire;Skovierova, Henrieta;Roberts, Mark
通讯作者：
Roberts, Mark
DOI：
10.1073/pnas.0806929106
发表时间：
2009-01-06
影响因子：
11.1
作者：
Deigan, Katherine E.;Li, Tian W.;Weeks, Kevin M.
通讯作者：
Weeks, Kevin M.