2017
DOI: 10.1128/jb.00656-16
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Characterization of Engineered PreQ1 Riboswitches for Inducible Gene Regulation in Mycobacteria

Abstract: We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis. U… Show more

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Cited by 14 publications
(13 citation statements)
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“…The preQ 1 -II riboswitch is a gene-regulatory ncRNA that attenuates translation by sensing cellular levels of preQ 1 (21,40,41). Translational regulation occurs by modulation of SDS accessibility-a common gene-regulation mechanism among bacterial riboswitches (1,9).…”
Section: Discussionmentioning
confidence: 99%
See 1 more Smart Citation
“…The preQ 1 -II riboswitch is a gene-regulatory ncRNA that attenuates translation by sensing cellular levels of preQ 1 (21,40,41). Translational regulation occurs by modulation of SDS accessibility-a common gene-regulation mechanism among bacterial riboswitches (1,9).…”
Section: Discussionmentioning
confidence: 99%
“…Using a similar approach, the Lrh preQ 1 -II riboswitch was used to control GFP expression in Mycobacterium, which does not possess queF genes required for preQ 1 biosynthesis ( Fig. 1A) (40). Considering the breadth of in vitro work on the preQ 1 -II riboswitch (21,25,(27)(28)(29)(30) and the success of GFPreporter systems for its functional analysis, we adapted the E. coli-based reporter assay to couple Lrh preQ 1 -II riboswitch regulation with GFPuv fluorescence, accompanied by orthogonal analysis of gene-regulatory conformations using in-cell chemical modification (41).…”
mentioning
confidence: 99%
“…Micklefield and coworkers used a rational targeted approach in the evaluation of synthetic compounds with riboswitch mutants and identified an orthogonal riboswitch−ligand pair that effectively repressed the transcription of selected genes in B. subtilis [42]. More recently, rationally engineered preQ 1 riboswitches have been applied for inducible gene regulation in mycobacteria [43]. …”
Section: Resultsmentioning
confidence: 99%
“…To demonstrate that the genetic knockout caused the observed phenotypes, there are several options for genetic complementation by restoration of gene expression, including plasmids that integrate at 2 different phage attachment sites and episomal plasmids with both lowlevel and high-level constitutive promoters [60][61][62]. There are also several inducible promoter systems and riboswitch platforms that can be used for genetic complementation or to perform depletion studies to study essential mycobacterial genes [63][64][65][66][67][68][69][70][71]. Essential mycobacterial genes are often studied as potential drug targets.…”
Section: Exploiting the Awesome Power Of Mycobacterial Geneticsmentioning
confidence: 99%