Coupled Changes in Translation and Transcription during Cobalamin-Dependent Regulation of btuB Expression in Escherichia coli

Abstract

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 inE. 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 btuBstart 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 thebtuB 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 ofbtuB 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.

Alternative oxygen‐dependent and oxygen‐independent ribonucleotide reductases in Streptomyces: cross‐regulation and physiological role in response to oxygen limitation

“…However, the effect of abolishing B12 synthesis on increasing transcription of the class Ia RNR genes may result from a rather different mechanism. Adenosylcobalamin is known to repress, primarily at the translational level, the expression of a variety of genes involved in B12 synthesis and transport and genes that use B12 as a cofactor (Richter-Dahlfors and Andersson, 1992;Nou and Kadner, 1998), by direct binding of B12 to a conserved RNA structural element, or B12-box, in the 5¢ untranslated region of the gene (reviewed in Vitreschak et al 2003). Rodionov et al (2003) identified B12 elements in the 5¢ untranslated regions of S. coelicolor genes determining B12-independent isozymes of methionine synthase and ribonucleotide reductase.…”

Do mRNAs act as direct sensors of small molecules to control their expression?

“…The cob operon of Salmonella typhimurium encodes the genes required for synthesis of cobalamin, and the btuB gene of both E. coli and S. typhimurium encodes a transporter for cobalamin. All have been shown to be repressed by cobalamin posttranscriptionally (8,9). Just 5Ј of the AUG for each gene is a RBS hairpin, and each gene has a ''B 12 box'' in a long untranslated leader sequence.…”

Ribo-attenuators: novel elements for reliable and modular riboswitch engineering

“…The rst advantage of the developed Ribo-attenuator system is its enabling of riboswitch controlled expression of a new gene of interest without the inclusion of a 5' fusion. Typically such fusions have been employed 19,20,21,22 because direct replacement of a riboswitch's ORF can degrade its functionality 18 (as demonstrated in Figure 3). However, re-engineering a riboswitch in this fashion gives rise to a protein with a large fusion, which can severely impact function.…”

“…Thus, substituting the original ORF with a new one on a`start codon for start codon' basis can nullify the desired riboswitch response to a given ligand 18 . To overcome this lack of modularity many studies have created fusions comprised of a riboswitch, the rst few hundred base pairs of its working ORF, and a gene of interest 19,20,21,22 . However, this approach fails in many circumstances as it can alter the gene's functionality, since many enzymes will not work with the inclusion of large 5' fusions 23,24,25,26,27,28 .…”