Laboratory of Biosystem Dynamics, Computational Systems Biology Research Group, Department of Signal Processing, Tampere University of Technology, FI-33101 Tampere, Finland.
Nucleic Acids Res. 2012 Sep 1;40(17):8472-83. doi: 10.1093/nar/gks583. Epub 2012 Jun 22.
In Escherichia coli, tetracycline prevents translation. When subject to tetracycline, E. coli express TetA to pump it out by a mechanism that is sensitive, while fairly independent of cellular metabolism. We constructed a target gene, PtetA-mRFP1-96BS, with a 96 MS2-GFP binding site array in a single-copy BAC vector, whose expression is controlled by the tetA promoter. We measured the in vivo kinetics of production of individual RNA molecules of the target gene as a function of inducer concentration and temperature. From the distributions of intervals between transcription events, we find that RNA production by PtetA is a sub-Poissonian process. Next, we infer the number and duration of the prominent sequential steps in transcription initiation by maximum likelihood estimation. Under full induction and at optimal temperature, we observe three major steps. We find that the kinetics of RNA production under the control of PtetA, including number and duration of the steps, varies with induction strength and temperature. The results are supported by a set of logical pairwise Kolmogorov-Smirnov tests. We conclude that the expression of TetA is controlled by a sequential mechanism that is robust, whereas sensitive to external signals.
在大肠杆菌中，四环素阻止翻译。当大肠杆菌受到四环素的影响时，会通过一种对细胞代谢相对独立但又相当敏感的机制表达 TetA 以将其泵出。我们构建了一个靶基因 PtetA-mRFP1-96BS，它在单个拷贝的 BAC 载体中带有 96 个 MS2-GFP 结合位点阵列，其表达受 tetA 启动子的控制。我们测量了目标基因单个 RNA 分子的体内产生动力学作为诱导剂浓度和温度的函数。从转录事件之间的间隔分布中，我们发现 PtetA 的 RNA 产生是一个亚泊松过程。接下来，我们通过最大似然估计推断转录起始中显著的顺序步骤的数量和持续时间。在完全诱导和最佳温度下，我们观察到三个主要步骤。我们发现，受 PtetA 控制的 RNA 产生动力学，包括步骤的数量和持续时间，随诱导强度和温度而变化。这一结果得到了一组逻辑成对的柯尔莫哥洛夫-斯米尔诺夫检验的支持。我们得出结论，TetA 的表达受一种顺序机制的控制，这种机制具有鲁棒性，但对外界信号敏感。
