A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae.
A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae.
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DOI：
10.1038/msb4100168
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发表时间：
2007
影响因子：
9.9
通讯作者：
中科院分区：
文献类型：
--
作者：
蛋白质降解速率由降解机制调节，降解机制清除不必要的管家蛋白并维持转录调节因子的适当动态分辨率。周转率也是至关重要的荧光报告，必须达到足够的荧光信号检测和时间分辨率跟踪动态响应之间的平衡。在这里，我们使用大肠杆菌降解机制的组成部分，以构建一个酿酒酵母菌株，允许可调降解的标记蛋白质。使用为单细胞荧光测量量身定制的微流体平台，我们使用ssrA标记的荧光报告基因监测蛋白质阻遏后的衰减率。我们观察到的半衰期范围为91至22分钟，这取决于降解基因的激活水平。计算建模的基础设置的酶促反应导致GFP的衰减曲线，是在非常好的协议与观察，这意味着降解是由Michaelis-Menten型相互作用。除了提供一个可调动态分辨率的报告，我们的研究结果为探索蛋白质降解对基因调控和信号通路的影响奠定了基础。
Protein decay rates are regulated by degradation machinery that clears unnecessary housekeeping proteins and maintains appropriate dynamic resolution for transcriptional regulators. Turnover rates are also crucial for fluorescence reporters that must strike a balance between sufficient fluorescence for signal detection and temporal resolution for tracking dynamic responses. Here, we use components of the Escherichia coli degradation machinery to construct a Saccharomyces cerevisiae strain that allows for tunable degradation of a tagged protein. Using a microfluidic platform tailored for single-cell fluorescence measurements, we monitor protein decay rates after repression using an ssrA-tagged fluorescent reporter. We observe a half-life ranging from 91 to 22 min, depending on the level of activation of the degradation genes. Computational modeling of the underlying set of enzymatic reactions leads to GFP decay curves that are in excellent agreement with the observations, implying that degradation is governed by Michaelis–Menten-type interactions. In addition to providing a reporter with tunable dynamic resolution, our findings set the stage for explorations of the effect of protein degradation on gene regulatory and signalling pathways.
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影响因子：
10.5
作者：
Gottesman, S;Roche, E;Sauer, RT
通讯作者：
Sauer, RT
影响因子：
3.5
作者：
van Dyck, L;Dembowski, M;Langer, T
通讯作者：
Langer, T
影响因子：
2.6
作者：
Sheff, MA;Thorn, KS
通讯作者：
Thorn, KS
DOI：
10.1073/pnas.0404733101
发表时间：
2004-08-17
影响因子：
11.1
作者：
Hersch, GL;Baker, TA;Sauer, RT
通讯作者：
Sauer, RT
影响因子：
64.8
作者：
Volfson, D;Marciniak, J;Hasty, J
通讯作者：
Hasty, J