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Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules

Babiskin, Andrew H. and Smolke, Christina D. (2011) Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules. Nucleic Acids Research, 39 (12). pp. 5299-5311. ISSN 0305-1048.

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The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast.

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Smolke, Christina D.0000-0002-5449-8495
Additional Information:© 2011 The Author(s). Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received December 29, 2010; Revised January 27, 2011; Accepted February 3, 2011. First published online: February 25, 2011. We thank K. Hoff for assistance in the expression and purification of Rnt1p; and S. Bastian and F.H. Arnold for assistance in sonication and FPLC. The National Science Foundation (CAREER award CBET-0917705 to C.D.S.); Alfred P. Sloan Foundation, fellowship (to C.D.S.). Funding for open access charge: National Science Foundation CBET-0917705.
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Alfred P. Sloan FoundationUNSPECIFIED
Issue or Number:12
Record Number:CaltechAUTHORS:20110801-082725157
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Official Citation:Andrew H. Babiskin and Christina D. Smolke Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules Nucl. Acids Res. (2011) 39(12): 5299-5311 doi:10.1093/nar/gkr090
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:24604
Deposited By: Ruth Sustaita
Deposited On:01 Aug 2011 15:49
Last Modified:03 Oct 2019 02:58

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