1887

Abstract

possesses interlinked routes for the synthesis of proline. The ProJ–ProA–ProH route is responsible for the production of proline as an osmoprotectant, and the ProB–ProA–ProI route provides proline for protein synthesis. We show here that the transcription of the anabolic and genes is controlled in response to proline limitation via a T-box-mediated termination/antitermination regulatory mechanism, a tRNA-responsive riboswitch. Primer extension analysis revealed mRNA leader transcripts of 270 and 269 nt for the and genes, respectively, both of which are synthesized from SigA-type promoters. These leader transcripts are predicted to fold into two mutually exclusive secondary mRNA structures, forming either a terminator or an antiterminator configuration. Northern blot analysis allowed the detection of both the leader and the full-length and transcripts. Assessment of the level of the transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a operon fusion reporter strain demonstrated that transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the and the leader sequences contain a CCU proline-specific specifier codon prone to interact with the corresponding uncharged proline-specific tRNA. By replacing the CCU proline specifier codon in the T-box leader with UUC, a codon recognized by a Phe-specific tRNA, we were able to synthetically re-engineer the proline-specific control of transcription to a control that was responsive to starvation for phenylalanine.

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2011-04-01
2019-10-23
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