title: T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis.
abstract: Bacillus subtilis 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 proBA and proI 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 proBA and proI 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 proBA and proI transcripts. Assessment of the level of the proBA transcripts revealed that the amount of the full-length mRNA species strongly increased in proline-starved cultures. Genetic studies with a proB-treA operon fusion reporter strain demonstrated that proBA transcription is sensitively tied to proline availability and is derepressed as soon as cellular starvation for proline sets in. Both the proBA and the proI 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 proBA 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 proBA transcription to a control that was responsive to starvation for phenylalanine.
authors.affiliation: Philipps-University Marburg, Department of Biology, Laboratory for Microbiology, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Brill
authors.affiliation: Philipps-University Marburg, Department of Biology, Laboratory for Microbiology, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany.
authors.forename: Tamara
authors.initials: T
authors.lastname: Hoffmann
authors.affiliation: CNRS UPR 9073 Insitut de Biologie Physico-Chimique (affiliated with Université de Paris 7 - Denis Diderot), 13 rue Pierre et Marie Curie, F-75005 Paris, France.
authors.forename: Harald
authors.initials: H
authors.lastname: Putzer
authors.affiliation: Philipps-University Marburg, Department of Biology, Laboratory for Microbiology, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany.
authors.forename: Erhard
authors.initials: E
authors.lastname: Bremer
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D053898:Biosynthetic Pathways; D015152:Blotting, Northern; D015964:Gene Expression Regulation, Bacterial; D009690:Nucleic Acid Conformation; D010649:Phenylalanine / Q000378:metabolism; D011392:Proline / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D011995:Recombination, Genetic; D058928:Riboswitch; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D058928:Riboswitch; D010649:Phenylalanine; D012343:RNA, Transfer; D011392:Proline