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首页> 外文期刊>Biochemistry >Mutations at Position-10 in the #lambda# P_R Promoter primarily Affect Conversion of the Initial Closed Complex (RP_c) to a Stable, Closed Intermediate (RP_i)
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Mutations at Position-10 in the #lambda# P_R Promoter primarily Affect Conversion of the Initial Closed Complex (RP_c) to a Stable, Closed Intermediate (RP_i)

机译：＃lambda＃P_R启动子中位置10的突变主要影响初始封闭复合物（RP_c）向稳定，封闭中间物（RP_i）的转化。

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The effects of mutations of -10 T:A to A:T, C:G, or G:C in the). PR promoter on formation of transcriptionally competent open complexes were studied by DNAse I footprinting, KMnO4-sensitivity, and abortive initiation kinetic analysis. The mutations -lOA (T:A -A:T) and -lOC significantly reduce kf, the composite rate constant for conversion of closed complexes (RPc) to open complexes (RPo) but do not affect KB, the equilibirium constant for formation of closed complexes. Unlike the other mutants or wild-type PR, the mutation with the largest effect on open complex formation, -lOG (T:A -G:C), substantially decreases the occupancy of the promoter. When reduced occupancy is taken into account, the calculated effect of the mutation on kf is a 20-fold reduction, Analysis of open complex formation by a three-step pathway that includes an additional intermediate, RPj, indicates that the primary effect of all three mutations is a reduction in the rate of isomerization of RP c to RPj, which precedes DNA strand separation. Thus, RNA polymerase holoenzyme must recognize specific base pairs in the -10 region of PR while the DNA is still double-stranded. Comparison of the observed level of stable complexes (RPj plus RP 0) with the level of productive complexes (RP 0) indicates that the -lOG mutation may also affect the equilibrium between RPj and RPo at 37°, Open complexes formed at the three mutant promoters are approximately 3-5 times less stable atJ37° than those formed at wild-type P_R.
机译：-10 T：A突变为A：T，C：G或G：C的影响）。通过DNAse I足迹，KMnO4敏感性和流产起始动力学分析研究了在转录感受态开放复合体形成过程中的PR启动子。突变-lOA（T：A -A：T）和-lOC显着降低kf，kf是封闭复合物（RPc）转化为开放复合物（RPo）的复合速率常数，但不影响KB（KB）的形成平衡常数。封闭的复合体。与其他突变体或野生型PR不同，对开放复合物形成影响最大的突变-10G（T：A -G：C）大大降低了启动子的占有率。当考虑减少的占用率时，突变对kf的计算影响为减少20倍。通过包括附加中间体RPj的三步途径对开放复合物形成的分析表明，这三者的主要作用突变是在DNA链分离之前RP c到RPj的异构化速率降低。因此，RNA聚合酶全酶必须识别PR的-10区中的特定碱基对，而DNA仍是双链的。将观察到的稳定复合物水平（RPj加RP 0）与生产性复合物水平（RP 0）进行比较表明，-10G突变也可能影响RPj和RPo在37°时的平衡，三个突变体形成开放复合物在J37°启动子的稳定性比在野生型P_R形成的启动子低约3-5倍。

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