Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

Science 335 (6065): 233-235

Copyright © 2012 by the American Association for the Advancement of Science

Widespread Genetic Switches and Toxicity Resistance Proteins for Fluoride

Jenny L. Baker1,*, Narasimhan Sudarsan2,3,*, Zasha Weinberg2,3, Adam Roth2,3, Randy B. Stockbridge4, and Ronald R. Breaker2,3,5,{dagger}

1 Department of Chemistry, Yale University, Box 208103, New Haven, CT 06520, USA.
2 Howard Hughes Medical Institute, Yale University, Box 208103, New Haven, CT 06520, USA.
3 Department of Molecular, Cellular, and Developmental Biology, Yale University, Box 208103, New Haven, CT 06520, USA.
4 Department of Biochemistry and Howard Hughes Medical Institute, Brandeis University, Waltham, MA 02454, USA.
5 Department of Molecular Biophysics and Biochemistry, Yale University, Box 208103, New Haven, CT 06520, USA.


Figure 1
View larger version (69K):
[in this window]
[in a new window]

 
Fig. 1. Fluoride binding by crcB motif RNAs. (A) Consensus sequence and structural model for crcB RNAs based on 2188 representatives from bacterial and archaeal species. P1, P2, P3 and pseudoknot labels identify extended base-paired substructures. (B) Sequence and secondary structure model for the WT 78 Psy RNA from P. syringae. Colored circles summarize the in-line probing results presented in (C). The two G residues preceding nucleotide 1 were added to facilitate RNA production. (C) Polyacrylamide gel electrophoresis analysis of an in-line probing assay with 78 Psy RNA and various amounts of fluoride. NR, T1, and -OH designate no reaction, partial digestion with RNase T1 (cleaves after guanosines), or partial digestion with hydroxide ions (cleaves after any nucleotide), respectively. Precursor RNA (Pre) band and some RNase T1 product bands are labeled (left). Locations of fluoride-mediated spontaneous RNA cleavage suppression (regions 1, 3, 5, 6) and enhancement (regions 2, 4) are identified by vertical bars. (D) Plot of the normalized fraction of RNA cleavage versus fluoride ion concentration from the data in (C). Curves represent those expected for one-to-one binding with a KD of 60 μM.

 

Figure 2
View larger version (40K):
[in this window]
[in a new window]

 
Fig. 2. Fluoride riboswitch-mediated gene control. (A) Solid media cultures of WT E. coli cells or crcB KO E. coli cells transformed with a riboswitch reporter fusion construct carrying the P. syringae eriC fluoride riboswitch. (B) Plot of the β-galactosidase reporter activity versus fluoride concentration (c) in liquid media supporting growth of transformed E. coli cells [see (A)] as quantified using Miller assays. WT and crcB KO E. coli cells grown in media supplemented with 50 mM NaCl (no added fluoride) yielded 0.06 and 15.5 Miller units, respectively.

 

Figure 3
View larger version (24K):
[in this window]
[in a new window]

 
Fig. 3. Evaluation of putative fluoride transport proteins. (A) Liquid cultures supplemented with specific amounts of NaF were inoculated with identical amounts of WT E. coli cells and the optical density (O.D.) at 600 nm was periodically recorded over a 16-hour period. (B) Growth curve plots for the E. coli crcB KO strain. (C) Anion efflux of an EriCF protein associated with the P. syringae fluoride riboswitch. Gray and black lines depict ion-transport measurements from liposomes in the absence or presence of protein, respectively. The high fluoride baseline is due to the relative membrane permeability of HF (pKa of 3.4) compared with chloride (pKa of –7). Asterisks in (C) and (D) identify the times at which protein-mediated anion transport is initiated. (D) Anion efflux by an EriC protein from E. coli that is known to serve as a chloride transporter.

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882