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Published October 2011 | Published + Supplemental Material
Journal Article Open

Synthetic RNA modules for fine-tuning gene expression levels in yeast by modulating RNase III activity


The design of synthetic gene networks requires an extensive genetic toolbox to control the activities and levels of protein components to achieve desired cellular functions. Recently, a novel class of RNA-based control modules, which act through post-transcriptional processing of transcripts by directed RNase III (Rnt1p) cleavage, were shown to provide predictable control over gene expression and unique properties for manipulating biological networks. Here, we increase the regulatory range of the Rnt1p control elements, by modifying a critical region for enzyme binding to its hairpin substrates, the binding stability box (BSB). We used a high throughput, cell-based selection strategy to screen a BSB library for sequences that exhibit low fluorescence and thus high Rnt1p processing efficiencies. Sixteen unique BSBs were identified that cover a range of protein expression levels, due to the ability of the sequences to affect the hairpin cleavage rate and to form active cleavable complexes with Rnt1p. We further demonstrated that the activity of synthetic Rnt1p hairpins can be rationally programmed by combining the synthetic BSBs with a set of sequences located within a different region of the hairpin that directly modulate cleavage rates, providing a modular assembly strategy for this class of RNA-based control elements.

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 (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received December 18, 2010; Revised April 5, 2011; Accepted May 15, 2011. Published online 6 July 2011. We thank J. Liang and A. Chang for assistance with FACS and for providing the pCS1585 and pCS1748 plasmids, K. Hoff for assistance in the expression and purification of Rnt1p and S. Bastian and F. H. Arnold for assistance in sonication and FPLC. Funding: 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). Conflict of interest statement. None declared.

Attached Files

Published - Babiskin2011p16293Nucleic_Acids_Res.pdf

Supplemental Material - Supplementary_Information.pdf


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