Nou X, Kadner R J
Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
J Bacteriol. 1998 Dec;180(24):6719-28. doi: 10.1128/JB.180.24.6719-6728.1998.
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.
在钴胺素存在的情况下生长，大肠杆菌外膜中维生素B12转运蛋白BtuB的水平会大幅降低。先前对调控突变体和btuB - lacZ融合体的分析表明，btuB基因调控的主要位点在翻译水平，这需要整个240个核苷酸（nt）的前导区序列。钴胺素依赖性转录融合体的调控程度较小，但除了前导区外，还需要编码序列前100 nt内的序列，称为翻译调控区（TRR）。为了分析大肠杆菌中btuB转录水平调控的过程，通过S1核酸酶保护试验分析了btuB RNA的水平和代谢，并分析了改变翻译和转录控制偶联的突变。发现转录融合体的表达与完整btuB RNA水平的变化相关，并且与通常寿命较长的RNA的代谢稳定性变化有关。突变分析表明，btuB起始密码子和一个可隔离Shine - Dalgarno序列的发夹结构对于钴胺素依赖性调控是必需的，并且TRR的翻译对于延长RNA稳定性和转录融合体的表达是必需的。转录起始阶段不存在调控这一点得到了以下发现的证实：几个截短的btuB RNA片段以组成型方式表达，并且即使btuB启动子和上游序列被异源bla和lac启动子取代，正常的调控反应仍然发生。由噬菌体T7 RNA聚合酶驱动的转录不受钴胺素调控，但在翻译水平上仍保留一些调控。从仅在钴胺素存在下产生且与调控结果无关的转录本片段的RNase III依赖性产生中，提示了钴胺素依赖性RNA结构变化。这些结果表明，钴胺素对btuB表达的主要控制发生在翻译起始水平，这在需要完整翻译调控区存在的过程中直接影响btuB RNA的水平和稳定性。
