Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach

Nucleic Acids Research
Kathrin LangRonald Micura

Abstract

Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain a...Continue Reading

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Citations

Jun 18, 2011·Accounts of Chemical Research·Andrea HallerRonald Micura
Oct 12, 2010·Biochemistry·Jinwei ZhangAdrian R Ferré-D'Amaré
Jul 6, 2010·Journal of the American Chemical Society·Kady-Ann SteenKevin M Weeks
Aug 3, 2012·Journal of the American Chemical Society·Kady-Ann SteenKevin M Weeks
May 3, 2011·Nature Chemical Biology·Andrea HallerRonald Micura
May 3, 2011·Nature Chemical Biology·Benoit HeppellDaniel A Lafontaine
May 19, 2011·Nature Chemical Biology·Ming C Hammond
Jun 11, 2010·Proceedings of the National Academy of Sciences of the United States of America·Ulrike RiederRonald Micura
Jan 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Peter C AnthonySteven M Block
Feb 27, 2013·Proceedings of the National Academy of Sciences of the United States of America·Andrea HallerRonald Micura
May 20, 2011·The Journal of Biological Chemistry·Jermaine L JenkinsJoseph E Wedekind
Nov 28, 2008·Nucleic Acids Research·Luc JaegerCody Geary
Oct 22, 2009·Nucleic Acids Research·Barbara PufferRonald Micura
Oct 11, 2011·Nucleic Acids Research·David AusländerMartin Fussenegger
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Methods Mentioned

BETA
fluorescence spectroscopy
for
X-ray

Software Mentioned

Synergy
Kaleidagraph

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