The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

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• 作者：Jillian Tyrrell ; Jennifer L. McGinnis ; Kevin M. Weeks ; Gary J. Pielak
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：December 3, 2013
• 年：2013
• 卷：52
• 期：48
• 页码：8777-8785
• 全文大小：451K
• 年卷期：v.52,no.48(December 3, 2013)
• ISSN：1520-4995

文摘

There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2鈥?hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg²⁺, the approximate Mg²⁺ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg²⁺ concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg²⁺ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.