Smith L D, Bertrand K P
Department of Microbiology and Molecular Genetics, University of California, Irvine 92717.
J Mol Biol. 1988 Oct 20;203(4):949-59. doi: 10.1016/0022-2836(88)90120-9.
Tetracycline induces transcription of the Tn10 tetracycline resistance gene (tetA) by binding to the tet repressor, thereby reducing the repressor's affinity for two operator sites that overlap the tet promoters. We characterized mutations in the tet repressor (tetRs mutations) that interfere with induction of tetA expression. The mutations were isolated on multicopy Tn10 tet plasmids by selecting for resistance to the inducer 5a,6-anhydrotetracycline. Under these conditions, maximal induction of tetA expression inhibits the growth of Escherichia coli K-12. DNA sequence analysis of 25 spontaneous tetRs mutations identified amino acid changes at 13 different positions clustered near the middle of the 207 amino acid residue sequence of tet repressor. This region (residues 64 to 107) is distinct from the bihelical DNA-binding motif of tet repressor (residues 26 to 47). The capacity of tetRs repressors to bind tet operator DNA and to respond to inducer was examined in vivo in tetA-lacZ fusion strains. In three cases, the capacity of tetRs repressors to bind tetracycline was examined in vitro using cell extracts enriched in repressor. Mutations 64Y (His64----Tyr) and 82H (Asn82----His) reduce the repressor's affinity for tetracycline more than 1000-fold and more than 100-fold, respectively, suggesting that His64 and Asn82 may be part of the inducer-binding site or directly involved in maintaining its conformation. Mutation 103I (Thr103----Ile) reduces the repressor's affinity for tetracycline less than tenfold, yet it interferes with induction to a greater extent than either 64Y or 82H, suggesting that 103I may also reduce the repressor's capacity to undergo a conformational change required for induction. The properties of tetRs mutants suggest that the region of amino acid residues 64 to 107 is involved in inducer binding and in signalling between the inducer-binding and operator-binding domains of the repressor.
四环素通过与四环素阻遏物结合来诱导Tn10四环素抗性基因（tetA）的转录，从而降低阻遏物对与tet启动子重叠的两个操纵位点的亲和力。我们对干扰tetA表达诱导的四环素阻遏物中的突变（tetRs突变）进行了表征。通过选择对诱导剂5a,6-脱水四环素的抗性，在多拷贝Tn10 tet质粒上分离出这些突变。在这些条件下，tetA表达的最大诱导会抑制大肠杆菌K-12的生长。对25个自发tetRs突变进行的DNA序列分析确定了在四环素阻遏物207个氨基酸残基序列中间附近的13个不同位置的氨基酸变化。该区域（第64至107位氨基酸残基）与四环素阻遏物的双螺旋DNA结合基序（第26至47位氨基酸残基）不同。在tetA-lacZ融合菌株中，在体内检测了tetRs阻遏物结合tet操纵子DNA和对诱导剂作出反应的能力。在三种情况下，使用富含阻遏物的细胞提取物在体外检测了tetRs阻遏物结合四环素的能力。突变64Y（His64→Tyr）和82H（Asn82→His）分别使阻遏物对四环素的亲和力降低了1000倍以上和100倍以上，这表明His64和Asn82可能是诱导剂结合位点的一部分或直接参与维持其构象。突变103I（Thr103→Ile）使阻遏物对四环素的亲和力降低不到10倍，但它比64Y或82H在更大程度上干扰诱导，这表明103I也可能降低阻遏物进行诱导所需构象变化的能力。tetRs突变体的特性表明，第64至107位氨基酸残基区域参与诱导剂结合以及阻遏物的诱导剂结合域与操纵子结合域之间的信号传递。
