TY - JOUR
T1 - Rational re-engineering of a transcriptional silencing PreQ1 riboswitch
AU - Wu, M.-C.
AU - Lowe, P.T.
AU - Robinson, C.J.
AU - Vincent, H.A.
AU - Dixon, N.
AU - Leigh, J.
AU - Micklefield, J.
N1 - This work was supported by BBSRC grant BB/I012648/1, Manchester Centre for Synthetic Biology (SynBioChem) BB/M017702/1, and a BBSRC Ph.D. studentship (for J.L.). The EPSRC provided a Ph.D. studentship (for P.T.L.).
PY - 2015/7/22
Y1 - 2015/7/22
N2 - 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-ligand 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. (Graph Presented).
AB - 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-ligand 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. (Graph Presented).
UR - http://pubs.acs.org/doi/suppl/10.1021/jacs.5b03405
U2 - 10.1021/jacs.5b03405
DO - 10.1021/jacs.5b03405
M3 - Article
AN - SCOPUS:84937683269
SN - 0002-7863
VL - 137
SP - 9015
EP - 9021
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 28
ER -