Betz J L, Fall M Z
Department of Biochemistry, Biophysics and Genetics, University of Colorado Medical School, Denver 80262.
Gene. 1988 Jul 30;67(2):147-58. doi: 10.1016/0378-1119(88)90392-7.
The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139-4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene. Six of the I-d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303-307], the His-18 repressor did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.
为了探究乳糖（lac）阻遏物与其靶标的序列特异性相互作用，研究了显性负性（I-d）lac阻遏物与野生型（wt）以及突变型（Oc）lac操纵基因的特异性结合。将编码N端60个氨基酸（aa）替换的突变型lacI基因克隆到质粒pBR322的衍生物中。其中12个lacI-d错义突变通过一般基因重组和分子克隆从F'lac附加体转移而来，9个lacI错义突变从M13-lacI噬菌体中重新克隆[莫特等人，《核酸研究》12（1984）4139 - 4152]。检测了突变型阻遏物的多肽大小和稳定性、与诱导剂异丙基-β-D-硫代半乳糖苷（IPTG）的结合以及与野生型操纵基因的结合。突变型阻遏物对野生型操纵基因的亲和力范围从检测不到到野生型阻遏物的约1%，并且突变型阻遏物对从染色体lacIq基因表达的阻遏物的反式显性作用各不相同。6种I-d阻遏物在体内部分降解。所有阻遏物与IPTG结合的亲和力与野生型阻遏物大致相同。对与野生型操纵基因具有显著亲和力或在假定的操纵基因识别螺旋（氨基酸17 - 25）中有替换的阻遏物，在体内检测了它们对一系列单一位点Oc操纵基因的亲和力。虽然甘氨酸-18、丝氨酸-18和亮氨酸-18替换的阻遏物对操纵基因第7位的特异性发生了改变[埃布赖特，《美国国家科学院院刊》83（1986）303 - 307]，但组氨酸-18阻遏物并未影响特异性。这一结果可能与组氨酸的侧链长度比其他氨基酸替换更长有关。
