Hawley D K, McClure W R
Proc Natl Acad Sci U S A. 1980 Nov;77(11):6381-5. doi: 10.1073/pnas.77.11.6381.
The in vitro initiation properties of the PR promoter of bacteriophage lambda and of a PR mutant, x3, were compared. Using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with RNA polymerase. These lags corresponded to the average times required for the formation of transcriptionally active open complexes. By measuring the lags at different RNA polymerase concentrations, we could separate open complex formation into two steps, based on a simple model in which the initial bimolecular association of free promoter and polymerase in a closed complex is followed by an isomerization to the open complex. The contribution of each step to the overall rate of open complex formation was quantitated for both promoters. We found that the x3 mutation, which is located in the -35 region of PR, resulted in a decrease in the association constant for the initial binding to the closed complex to 5% of its wild-type value and a decrease in the rate of the isomerization to 20%. The lifetimes and abortive initiation characteristics of the mutant and wild-type promoters were similar. We concluded that the main effect of the x3 mutation was to increase the average time of open complex formation and that the functional properties of the open complexes did not differ significantly between the two promoters.
对噬菌体λ的PR启动子及其一个PR突变体x3的体外起始特性进行了比较。利用流产起始反应，我们测量了用RNA聚合酶起始二核苷酸合成时，达到最终稳态速率过程中的延迟。这些延迟对应于形成转录活性开放复合物所需的平均时间。通过测量不同RNA聚合酶浓度下的延迟，基于一个简单模型，我们可以将开放复合物的形成分为两个步骤，在该模型中，游离启动子和聚合酶在封闭复合物中的初始双分子结合之后是向开放复合物的异构化。对两个启动子而言，都对每个步骤对开放复合物形成总速率的贡献进行了定量。我们发现，位于PR的 -35区域的x3突变导致与封闭复合物初始结合的结合常数降至其野生型值的5%，异构化速率降至20%。突变型和野生型启动子的寿命及流产起始特性相似。我们得出结论，x3突变的主要影响是增加了开放复合物形成的平均时间，并且两个启动子的开放复合物功能特性没有显著差异。
