PROMOTER MUTATIONS AFFECTING DIVERGENT TRANSCRIPTION IN THE TN10 TETRACYCLINE RESISTANCE DETERMINANT
PROMOTER MUTATIONS AFFECTING DIVERGENT TRANSCRIPTION IN THE TN10 TETRACYCLINE RESISTANCE DETERMINANT
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DOI：
10.1016/0022-2836(85)90306-7
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发表时间：
1985-01-01
影响因子：
5.6
通讯作者：
BERTRAND, KP
中科院分区：
文献类型：
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作者：
DANIELS, DW;BERTRAND, KP
转座子Tn10中的四环素抗性决定基因由两个基因组成，即Teta抗性基因和TetR抑制基因，它们是从发散重叠的启动子转录而来的。我们测定了在多拷贝质粒上携带Tn10tet基因的大肠杆菌中脉冲标记的tet信使RNA的水平。加入诱导剂5a，6-脱水四环素后，TetA基因的表达增加260-430倍，TetR基因的表达增加35-65倍。根据在最大诱导条件下合成的TetA和TetR的相对摩尔数来判断，TetA启动子(ETPA)的活性是两个TetR启动子(TETPR1和TETPR2)的总和的7~11倍。我们鉴定了10个基因突变，其中包括9个单碱基对替换和1个30碱基对缺失。所有的单碱基改变都降低了与E.ColiRNA聚合酶识别的启动子的一致序列的一致性。在体内，高度保守核苷酸的突变会导致ETPA活性降低200到600倍。令人意想不到的是，尽管这些基因突变位于TETPR1和TETPR2的-35和-10区域之外，但它们在体内的结合活性却降低了两到四倍。在TetR-TetPR-LacZ操纵子融合菌株中，对于两个TetPA突变，也表现出对TetPR活性的负面影响，从而排除了由于质粒拷贝数或诱导效率的差异而导致的可能性。根据重叠的tet启动子竞争RNA聚合酶的预期，讨论了etPA突变的多效性效应。
The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping, promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 260- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally induced conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutation reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.