A quantitative assay for bacterial RNA polymerases.
A quantitative assay for bacterial RNA polymerases.
复制标题
细菌 RNA 聚合酶的定量测定。
DOI：
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发表时间：
1979
影响因子：
4.8
通讯作者：
N. Neff
中科院分区：
文献类型：
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作者：
M. Chamberlin;W. Nierman;J. Wiggs;N. Neff
本文设计了一种简单的方法，可以定量测定细菌RNA聚合酶制剂中活性酶的浓度。该方法还给出了几个参数的值，这些参数提供了RNA聚合酶在转录循环的不同单个步骤中的性质的定量描述，包括1）启动子位点定位加链起始的速率，2）链延伸的平均速率，和3）在特定转录终止信号下链终止的效率。该方法的原理取决于T '7 DNA的简单转录特性;在适当的条件下，仅使用单个转录单位，并且可以测量单轮转录中核糖核苷酸掺入的速率和程度。该方法已被校准与大肠杆菌RNA聚合酶全酶，有良好的定量信息，在转录的T7 DNA模板的不同步骤中的酶的行为，并为其中的活性酶的浓度可以通过末端标记程序独立地确定。当该程序被应用到一些不同的制备均匀的E。大肠杆菌全酶中，发现有活性的RNA聚合酶蛋白质的分数在很宽的范围内变化（10 - 90%），并且在这些制剂中T7终止子信号的利用效率也存在显著差异。相比之下，启动子选择加上链起始和链延长的速率对于这些不同的制剂似乎是相同的。鉴于这些巨大的差异，可能存在于不同的E。大肠杆菌RNA聚合酶，似乎重要的是，用于生物化学研究的制剂，仔细表征不同的活性参数，采用这种方法或一些等效的。该测定方法所基于的假设在大多数情况下似乎对来自广泛的分类学上不同的细菌菌株的细菌RNA聚合酶也是有效的。这是因为观察到这些不同的细菌RNA聚合酶都选择性地转录T7 DNA，并且通常使用与大肠杆菌相同的启动子和终止子位点。大肠杆菌酶（Wiggs，J.，Bush，J.，和Chamberlin，M. J.（1979）Cell 16，97 -109）。来自红色红孢菌、耻垢分枝杆菌、新月柄杆菌、棕色固氮菌和枯草芽孢杆菌的RNA聚合酶对T7 DNA的单循环转录曲线与E.大肠杆菌聚合酶，虽然
A simple procedure has been devised which permits the quantitative determination of the concentration of active enzyme in preparations of bacterial RNA polymerases. The procedure also gives values for several parameters which provide a quantitative description of the properties of the RNA polymerase in different individual steps of the transcription cycle including 1) the rate of promoter site location plus chain initiation, 2) the mean rate of chain elongation, and 3) the efficiency of chain termination at a specific transcriptional termination signal. The principle of the method depends on the simple transcriptional properties of T’7 DNA; under appropriate conditions only a single transcriptional unit is utilized and the rate and extent of ribonucleotide incorporation in a single round of transcription can be measured. The method has been calibrated with the Escherichia coli RNA polymerase holoenzyme for which there is good quantitative information as to the behavior of the enzyme in the different steps in transcription of a T7 DNA template, and for which the concentration of active enzyme can be determined independently by end-labeling procedures. When the procedure is applied to a number of different preparations of homogeneous E. coli holoenzyme, it is found that the fraction of RNA polymerase protein which is active varies over a wide range (10 to 90%), and that there are significant differences among these preparations in the efficiency of utilization of the T7 terminator signal as well. By contrast, the rates of promoter selection plus chain initiation and of chain elongation appear the same for these different preparations. Given these large differences that can exist in different preparations of the E. coli RNA polymerase, it seems important that preparations employed for biochemical studies be carefully characterized for the different activity parameters employing this method or some equivalent. The assumptions on which the assay procedure is based also appear to be valid in most cases for bacterial RNA polymerases from a wide range of taxonomically different bacterial strains. This follows from the observation that these diverse bacterial RNA polymerases all transcribe T7 DNA selectively, and generally employ the same promoter and terminator sites used by the E. coli enzyme (Wiggs, J., Bush, J., and Chamberlin, M. J. (1979) Cell 16,97-109). RNA polymerases from Rhodosporillum rubrum, Mycobacterium smegmatis, Caulobacter crescentus, Azotobacter vinelandii, and Bacillus subtilis give single cycle transcriptional curves with T7 DNA similar to those of the E. coli polymerase, although