Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states
Reconciling molecular regulatory mechanisms with noise patterns of bacterial metabolic promoters in induced and repressed states
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DOI：
10.1073/pnas.1110541108
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发表时间：
2012-01-03
影响因子：
11.1
通讯作者：
Royer, Catherine A.
中科院分区：
文献类型：
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作者：
Ferguson, Matthew L.;Le Coq, Dominique;Royer, Catherine A.
评估基因表达噪声以获得机理见解需要在大的动态范围内精确定量许多单个细胞上的基因表达。我们使用了一种独特的方法，基于双光子荧光波动显微镜直接测量，在单细胞水平和单分子的灵敏度，荧光蛋白的绝对浓度产生的两个枯草芽孢杆菌启动子，控制糖酵解和酵母异生之间的开关。我们量化了生长在葡萄糖或苹果酸盐上的报告菌株中GFP浓度的细胞间变化，包括在强烈的分解代谢物抑制下非常弱的转录基因。结果显示强烈的转录爆发，特别是糖酵解启动子。噪声模式参数的两个拮抗性启动子控制的营养开关的糖酵解和产气碳源的差异影响，区分不同的机制，控制他们的活动。我们的转录事件的随机模型再现了所观察到的噪声模式，并确定了负责启动子表达谱差异的关键参数。该模型还解决了这些启动子的体外操纵子亲和力和体内阻遏物活性之间的明显矛盾。最后，我们的研究结果表明，负反馈是没有噪音减少的情况下，强烈的转录爆发。
Assessing gene expression noise in order to obtain mechanistic insights requires accurate quantification of gene expression on many individual cells over a large dynamic range. We used a unique method based on 2-photon fluorescence fluctuation microscopy to measure directly, at the single cell level and with single-molecule sensitivity, the absolute concentration of fluorescent proteins produced from the two Bacillus subtilis promoters that control the switch between glycolysis and gluconeogenesis. We quantified cell-to-cell variations in GFP concentrations in reporter strains grown on glucose or malate, including very weakly transcribed genes under strong catabolite repression. Results revealed strong transcriptional bursting, particularly for the glycolytic promoter. Noise pattern parameters of the two antagonistic promoters controlling the nutrient switch were differentially affected on glycolytic and gluconeogenic carbon sources, discriminating between the different mechanisms that control their activity. Our stochastic model for the transcription events reproduced the observed noise patterns and identified the critical parameters responsible for the differences in expression profiles of the promoters. The model also resolved apparent contradictions between in vitro operator affinity and in vivo repressor activity at these promoters. Finally, our results demonstrate that negative feedback is not noise-reducing in the case of strong transcriptional bursting.