Martin Robert G, Rosner Judah L
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bldg. 5, Rm 333, National Institutes of Health, Bethesda, MD 20892-0560, USA.
Mol Microbiol. 2004 Jul;53(1):183-91. doi: 10.1111/j.1365-2958.2004.04080.x.
The marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the -35 and -10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild-type MarR. When both sites were wild type, transcription in the derepressed marR-deleted strain was 19-fold that of the wild-type strain; when only site I or site II was wild type, this ratio was reduced to 4.3- and 2.6-fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild-type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine-Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem-loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild-type strain. However, a single basepair mutation that strengthens the stem-loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5 bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50-75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.
大肠杆菌的marRAB多抗生素抗性操纵子受MarR自动阻遏。MarR在体外与两个回文序列结合：位点I位于RNA聚合酶结合的 -35和 -10六聚体之间并与之重叠；位点II位于marR的转录起始位点和GTG起始密码子之间。为了评估这些位点在体内的重要性，在有和没有野生型MarR的情况下，使用与lacZ的融合体分析了突变位点对转录的影响。当两个位点均为野生型时，去阻遏的marR缺失菌株中的转录是野生型菌株的19倍；当只有位点I或位点II是野生型时，该比率分别降至4.3倍和2.6倍，表明完全阻遏需要两个位点，但一个位点也可以独立于另一个位点发生一些阻遏。野生型启动子与lacZ的翻译融合表明，marR的翻译仅以转录速率的4.5%进行。对具有突变前导序列的翻译融合体的分析表明，翻译效率低下的主要原因是Shine-Dalgarno（SD）序列AGG(G)较弱。尽管SD序列位于位点II的潜在茎环结构内，但在野生型菌株中未发现SD序列被封闭的证据。然而，一个增强茎环结构的单碱基对突变极大地降低了翻译效率。将起始密码子从GTG替换为ATG可使翻译效率提高50%。将SD序列与GTG密码子之间的5 bp间距增加1至4个碱基可使翻译效率降低50-75%。marR的低效翻译可能有助于使细胞对环境信号敏感。
