Coupled changes in translation and transcription during cobalamin-dependent regulation of btuB expression in...

The level of the vitamin B12 transport protein BtuB in the outer membrane of Escherichia coli is strongly reduced by growth in the presence of cobalamins. Previous analyses of regulatory mutants and of btuB-lacZ fusions indicated that the primary site of btuB gene regulation was at the translational level, and this required sequences throughout the 240-nucleotide (nt) leader region. Cobalamin-dependent regulation of transcriptional fusions was of a lesser magnitude but required, in addition to the leader, sequences within the first 100 nt of the coding sequence, termed the translated regulatory region (TRR). To analyze the process of transcription-level regulation of btuB in E. coli, the levels and metabolism of btuB RNA were analyzed by S1 nuclease protection assays, and mutations that alter the coupling of translational and transcriptional control were analyzed. Expression of transcriptional fusions was found to correlate with changes in the level of intact btuB RNA and was related to changes in the metabolic stability of the normally long-lived RNA. Mutational analysis showed that the btuB start codon and a hairpin structure that can sequester the Shine-Dalgarno sequence are necessary for cobalamin-dependent regulation and that translation of the TRR is necessary for extended RNA stability and for expression of the transcriptional fusion. The absence of regulation at the stage of transcription initiation was confirmed by the findings that several truncated btuB RNA fragments were expressed in a constitutive manner and that the normal regulatory response occurred even when the btuB promoter and upstream sequences were replaced by the heterologous bla and lac promoters. Transcription driven by phage T7 RNA polymerase was not regulated by cobalamins, although some regulation at the translational level was retained. Cobalamin-dependent changes in RNA structure were suggested from the RNase III-dependent production of a transcript fragment that is made only in the presence of cobalamin and is independent of the regulatory outcome. These results indicate that the primary control of btuB expression by cobalamin occurs at the level of translation initiation, which directly affects the level and stability of btuB RNA in a process that requires the presence of the intact translated regulatory region.