Fong R S, Woody S, Gussin G N
Biology Department, University of Iowa, Iowa City 52242.
J Mol Biol. 1993 Aug 5;232(3):792-804. doi: 10.1006/jmbi.1993.1432.
When the transcription startsites of the phage lambda promoters PRM and PR are separated by 82 bp (the wild-type spacing), mutating PR increases the rate of open complex formation at PRM at all RNA polymerase (RNAP) concentrations tested in vitro. This is reflected in a fourfold increase in kappa f (the rate constant for isomerization of closed to open complexes) and a threefold decrease in KB (the equilibrium constant for formation of closed complexes). These effects of mutating PR resemble qualitatively those we observed when the separation between the two promoters was decreased by a single base-pair, but are quantitatively less dramatic. Although mutating PR has the additional effect of uncovering a weak promoter, P alpha, which overlaps both PRM and PR, the presence of P alpha does not account for the effects of PR mutations on open complex formation at PRM. In fixed-time assays at a single RNAP concentration, repressor stimulated PRM approximately threefold on a PR- template, indicating that activation is mediated substantially by a direct interaction between repressor and RNAP. That is, activation of PRM is not merely an indirect consequence of repressing PR. Kinetic data confirm this conclusion. In a PR- genetic background, repressor increased kappa f six- to eightfold and decreased KB approximately twofold. Similar results were obtained when OR3 was mutated, indicating that the effect on KB is not due to repressor binding to OR3. Thus, repressor causes a significant increase in the rate of open complex formation at PRM even when PR is inactive. On a PR+ template, 75 nM repressor stimulated PRM by increasing kappa f eightfold, with no effect on KB, which agrees with previous results. However, increased repressor concentrations stimulated kappa f by an additional factor of two to four, indicating that previous experiments underestimated the effect of repressor on kappa f. At the same time, increasing the repressor concentration decreased KB for PRM on a wild-type template. At the highest repressor concentration tested (275 nM), KB decreased 15-fold, presumably due to OR3-mediated repression of PRM. However, at an intermediate repressor concentration (170 nM) values of kappa f and KB for PRM on a PR+ template were in close agreement with the corresponding parameters obtained on a PR- template. These data lead us to suggest that repressor causes a decrease in KB for PRM on both a PR+ and a PR- template independent of its ability to bind to OR3.
当噬菌体λ启动子PRM和PR的转录起始位点相隔82个碱基对（野生型间距）时，在体外测试的所有RNA聚合酶（RNAP）浓度下，突变PR都会提高PRM处开放复合物形成的速率。这表现为κf（从封闭复合物异构化为开放复合物的速率常数）增加四倍，KB（封闭复合物形成的平衡常数）降低三倍。突变PR的这些效应在定性上类似于我们观察到的当两个启动子之间的间距减少一个碱基对时的情况，但在定量上不那么显著。尽管突变PR还有揭示一个弱启动子Pα的额外效应，Pα与PRM和PR都重叠，但Pα的存在并不能解释PR突变对PRM处开放复合物形成的影响。在单一RNAP浓度下的定时实验中，阻遏物在PR -模板上对PRM的刺激约为三倍，这表明激活主要是由阻遏物与RNAP之间的直接相互作用介导的。也就是说，PRM的激活不仅仅是抑制PR的间接结果。动力学数据证实了这一结论。在PR -遗传背景下，阻遏物使κf增加六至八倍，KB降低约两倍。当OR3突变时也获得了类似的结果，这表明对KB的影响不是由于阻遏物与OR3结合。因此，即使PR无活性，阻遏物也会导致PRM处开放复合物形成速率显著增加。在PR +模板上，75 nM阻遏物通过使κf增加八倍来刺激PRM，对KB没有影响，这与先前的结果一致。然而，阻遏物浓度增加会使κf额外增加两至四倍，这表明先前的实验低估了阻遏物对κf的影响。同时，增加阻遏物浓度会降低野生型模板上PRM的KB。在测试的最高阻遏物浓度（275 nM）下，KB降低了15倍，可能是由于OR3介导的对PRM的抑制。然而，在中等阻遏物浓度（170 nM）下，PR +模板上PRM的κf和KB值与在PR -模板上获得的相应参数非常一致。这些数据使我们认为，阻遏物在PR +和PR -模板上都会导致PRM的KB降低，这与其结合OR3的能力无关。
