Burz D S, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1994 Jul 19;33(28):8406-16. doi: 10.1021/bi00194a004.
Wild-type cI repressor dimers bind with 2.5-3 kcal/mol of cooperative free energy to the tripartite right operator region (OR) of bacteriophage lambda [Johnson, A. D., et al. (1981) Nature 294, 217-223; Brenowitz, M., et al. (1986) Methods Enzymol. 130, 132-181]. Quantitative modeling has suggested that cooperativity is required for maintenance of the lysogenic state and for the efficient switch from lysogenic to lytic growth [Ackers, G. K., et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1129-1133; Shea, M. A., & Ackers, G. K. (1985) J. Mol. Biol. 181, 211-230]. Cooperativity and self-association are thought to involve protein-protein contacts between C-terminal domains of the repressor molecule [Pabo, C. O., et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1608-1612]. To address the importance of the C-terminal domain in mediating the cooperativity exhibited by lambda cI repressor, a number of single-site mutant candidates were screened for possible deficiencies in cooperative interactions [Beckett, D., et al. (1993) Biochemistry 32, 9073-9079; Burz, D. S., et al. (1994) Biochemistry 33, 8399-8405]. Since repressor dimerization and binding to operator sites are coupled processes, elucidation of the energetic basis of regulation in this system requires that the equilibrium dimerization constants and the intrinsic and cooperative free energies of binding be measured. In this work we evaluate the interaction of eight mutant repressors with OR DNA: Gly147-->Asp (GD147), Pro158-->Thr (PT158), Glu188-->Lys (EK188), Lys192-->Asn (KN192), Tyr210-->His (YH210), Ser228-->Arg (SR228), and Ser228-->Asn (SN228), each with an amino acid substitution in the C-terminal domain, and Glu102-->Lys (EK102) where the substitution lies in the "linker sequence" between domains. Self-assembly properties of six of these mutant repressors are presented in the preceding paper (Burz et al., 1994). In this work, the binding of mutant cI repressors to OR was examined using quantitative DNAse I footprinting. This technique monitors individual site occupancy concurrent with binding at the other sites within a multisite complex.(ABSTRACT TRUNCATED AT 400 WORDS)
野生型cI阻遏蛋白二聚体以2.5 - 3千卡/摩尔的协同自由能与噬菌体λ的三方右操纵子区域（OR）结合[约翰逊，A.D.等人（1981年）《自然》294，217 - 223；布雷诺维茨，M.等人（1986年）《酶学方法》130，132 - 181]。定量建模表明，协同作用对于维持溶原状态以及从溶原生长向裂解生长的有效转变是必需的[阿克斯，G.K.等人（1982年）《美国国家科学院院刊》79，1129 - 1133；谢伊，M.A.和阿克斯，G.K.（1985年）《分子生物学杂志》181，211 - 230]。协同作用和自组装被认为涉及阻遏蛋白分子C端结构域之间的蛋白质 - 蛋白质接触[帕博，C.O.等人（1979年）《美国国家科学院院刊》76，1608 - 1612]。为了研究C端结构域在介导λ cI阻遏蛋白所表现出的协同作用中的重要性，筛选了许多单点突变候选物，以寻找协同相互作用中可能存在的缺陷[贝克特，D.等人（1993年）《生物化学》32，9073 - 9079；布尔兹，D.S.等人（1994年）《生物化学》33，8399 - 8405]。由于阻遏蛋白二聚化和与操纵子位点的结合是耦合过程，阐明该系统中调控的能量基础需要测量平衡二聚化常数以及结合的内在自由能和协同自由能。在这项工作中，我们评估了八个突变阻遏蛋白与OR DNA的相互作用：甘氨酸147→天冬氨酸（GD147）、脯氨酸158→苏氨酸（PT158）、谷氨酸188→赖氨酸（EK188）、赖氨酸192→天冬酰胺（KN192）、酪氨酸210→组氨酸（YH210）、丝氨酸228→精氨酸（SR228）和丝氨酸228→天冬酰胺（SN228），每个突变体在C端结构域都有一个氨基酸取代，还有谷氨酸102→赖氨酸（EK102），其取代位于结构域之间的“连接序列”中。前一篇论文（布尔兹等人，1994年）介绍了其中六个突变阻遏蛋白的自组装特性。在这项工作中，使用定量DNA酶I足迹法检测了突变cI阻遏蛋白与OR的结合。该技术在监测多位点复合物中其他位点结合的同时，监测单个位点的占据情况。（摘要截断于400字）
