Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch

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• 作者：Ming-Cheng Wu ; Phillip T. Lowe ; Christopher J. Robinson ; Helen A. Vincent ; Neil Dixon ; James Leigh ; Jason Micklefield
• 刊名：Journal of the American Chemical Society
• 出版年：2015
• 出版时间：July 22, 2015
• 年：2015
• 卷：137
• 期：28
• 页码：9015-9021
• 全文大小：450K
• ISSN：1520-5126

文摘

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ₁ riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch鈥搇igand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.