Hwang J J, Brown S, Gussin G N
Department of Biology, University of Iowa, Iowa City 52242.
J Mol Biol. 1988 Apr 20;200(4):695-708. doi: 10.1016/0022-2836(88)90481-0.
The mutation, prmE37, located at -14 in the PRM promoter of bacteriophage lambda, reduces PRM function dramatically both in vitro and in vivo. In a search for second-site revertants of prmE37, we isolated a double mutant that exhibits a partially restored Prm+ phenotype. The second-site mutation (at -31) is identical to the mutation prmup-1. The activity of the doubly mutant (pseudo-revertant) promoter, prmE37prmup-1, was investigated in vivo using a PRM-lacZ fusion phage and found to be intermediate between that of prmE37 and wild-type PRM. However, the relative strength of the prmE37prmup-1 promoter was greater than expected following superinfection of a lambda lysogen. Since nalidixic acid was found to preferentially inhibit transcription from the doubly mutant promoter under these conditions, we suggest that DNA supercoiling favors activation of this promoter by repressor. In runoff transcription assays in the absence of repressor, the activity of wild-type PRM and the doubly mutant promoter were the same. However, while addition of repressor significantly stimulated wild-type PRM, it had little or no effect on the activity of the doubly mutant promoter. Values of KB, the equilibrium constant for formation of closed complexes, and kf, the rate constant for isomerization of closed to open complexes, were determined in abortive initiation assays, and the product of kfKB was used as a measure of promoter strength. The results of these assays are in agreement with those obtained in runoff transcription assays. In the absence of repressor, values of kfKB for the doubly mutant promoter and wild-type PRM are the same; however, tau obs, the time required for open complex formation, is significantly greater for the double mutant than for wild-type PRM at all RNA polymerase concentrations used for the abortive initiation analysis. In the presence of repressor, the doubly mutant promoter is stronger than the prmE37 promoter, but much weaker than wild-type PRM. This is due to the fact that kf for the doubly mutant promoter is increased 2.5-fold by repressor, but KB is reduced to the same extent. These two effects counteract each other, so that repressor has no net effect on the strength of the prmE37prmup-1 promoter in vitro. In contrast, repressor increases kf for wild-type PRM eightfold and increases overall promoter strength (KBkf) nearly fivefold. In the presence of repressor, the effects of the two mutations, prmE37 and prmup-1, on kf are independent. This observation is discussed in relation to revised models for open complex formation.
噬菌体λ的PRM启动子中位于-14位置的突变prmE37，在体外和体内均显著降低PRM功能。在寻找prmE37的第二位点回复突变体的过程中，我们分离出了一个双突变体，其表现出部分恢复的Prm+表型。第二位点突变（位于-31）与突变prmup-1相同。使用PRM-lacZ融合噬菌体在体内研究了双突变（假回复）启动子prmE37prmup-1的活性，发现其活性介于prmE37和野生型PRM之间。然而，在λ溶原菌进行超感染后，prmE37prmup-1启动子的相对强度大于预期。由于发现在这些条件下萘啶酸优先抑制来自双突变启动子的转录，我们认为DNA超螺旋有利于阻遏物对该启动子的激活。在无阻遏物的径流转录分析中，野生型PRM和双突变启动子的活性相同。然而，添加阻遏物显著刺激野生型PRM时，对双突变启动子的活性几乎没有影响。在流产起始分析中测定了形成封闭复合物的平衡常数KB和封闭复合物异构化为开放复合物的速率常数kf，并将kfKB的乘积用作启动子强度的度量。这些分析结果与径流转录分析中获得的结果一致。在无阻遏物时，双突变启动子和野生型PRM的kfKB值相同；然而，在用于流产起始分析的所有RNA聚合酶浓度下，双突变体形成开放复合物所需的时间tau obs比野生型PRM长得多。在有阻遏物时，双突变启动子比prmE37启动子更强，但比野生型PRM弱得多。这是因为阻遏物使双突变启动子的kf增加了2.5倍，但KB降低了相同程度。这两种效应相互抵消，因此阻遏物在体外对prmE37prmup-1启动子的强度没有净影响。相比之下，阻遏物使野生型PRM的kf增加了八倍，并使整体启动子强度（KBkf）增加了近五倍。在有阻遏物时，prmE37和prmup-1这两个突变对kf的影响是独立的。结合开放复合物形成的修订模型对这一观察结果进行了讨论。
