title: Translational attenuation of the Bacillus subtilis spo0B cistron by an RNA structure encompassing the initiation region.
abstract: The spo0B gene, which exists as an operon with the obg gene, is required to initiate sporulation (stage 0) of Bacillus subtilis . This gene encodes a phosphotransferase in the multicomponent phosphorelay system. We here report the novel finding that a spo0B 5'-terminal SLR (stem-loop structure sequestering ribosome binding sequence; ACUCCUAA-X16-UUG GGAG U, Delta G = -8.71 kcal/mol) attenuated spo0B translation. The spo0B gene was efficiently transcribed but Spo0B protein was not overproduced in Escherichia coli when spo0B was induced using expression vectors carrying the SLR- spo0B region under control of the tac promoter. Deletion of the SLR from the vectors resulted in overexpression of spo0B . Therefore, to characterize expression of spo0B with a SLR in B.subtilis we constructed transcriptional and translational lacZ fusions combined with the spo0B 5'-terminal region with a deleted or mutagenized SLR. These constructs were subsequently introduced into B.subtilis as multiple and single copies, then beta-galactosidase activities were measured. The possible SLR also functioned as a negative cis element in B.subtilis. Furthermore, B.subtilis strain 1S16 (spo0B136) lysogenized straight phiCD0B-S and -W, harboring spo0B with mutagenized SLRs that were more (Delta G = -14.0 kcal/mol) and less-stable (Delta G = -1.31 kcal/mol) compared with the wild-type, exhibited null and wild-type sporulation respectively. These results indicate that the spo0B 5'-SLR affects spo0B gene expression for sporulation but that low expression of spo0B through the wild-type SLR was sufficient to initiate sporulation in B.subtilis.
authors.affiliation: Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu 183, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Asayama
authors.forename: K i
authors.initials: Ki
authors.lastname: Saito
authors.forename: Y
authors.initials: Y
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009876:Operon / Q000235:genetics; D017354:Point Mutation; D014176:Protein Biosynthesis / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D012808:Sigma Factor; D013171:Spores, Bacterial / Q000235:genetics; D014157:Transcription Factors
chemical_list: D001426:Bacterial Proteins; C090542:Obg GTP-binding protein, Bacteria; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; D014157:Transcription Factors; C092468:spoIIR protein, Bacillus subtilis; C030069:spore-specific proteins, Bacillus; D019204:GTP-Binding Proteins