Whole-cell modeling of E. coli confirms that in vitro tRNA aminoacylation measurements are insufficient to support cell growth and predicts a positive feedback mechanism regulating arginine biosynthesis.
Whole-cell modeling of E. coli confirms that in vitro tRNA aminoacylation measurements are insufficient to support cell growth and predicts a positive feedback mechanism regulating arginine biosynthesis.
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DOI：
10.1093/nar/gkad435
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发表时间：
2023-07-07
影响因子：
14.9
通讯作者：
中科院分区：
文献类型：
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作者：
在大肠杆菌中，体外tRNA氨酰化测量与体内蛋白质合成要求之间的不一致在近40年前被假设，但已被证明很难证实。通过提供体内细胞过程的整体表示，全细胞建模可以测试细胞在体外测量的参数化时是否以生理上正确的方式运行。在这里，tRNA氨酰化、基于密码子的多肽延长和N-末端蛋氨酸切割的机制模型被结合到正在开发的大肠杆菌全细胞模型中。随后的分析证实了氨基酰-tRNA合成酶动力学测量不足以维持细胞蛋白质组，并估计氨基酰-tRNA合成酶kcat的平均水平高出7.6倍。用惊慌失措的土猫模拟细胞生长，证明了这些体外测量对细胞表型的全球影响。例如，HisRS的kcat不足导致蛋白质合成对单细胞中氨酰-tRNA合成酶表达的自然变异性不那么强健。更令人惊讶的是，由于N-乙酰谷氨酸合成酶表达不足，ArgRS活性不足导致精氨酸生物合成受到灾难性影响，N-乙酰谷氨酸合成酶的翻译依赖于重复的CGG密码子。总体而言，扩展的E.Coli模型加深了对翻译如何在活体环境中运行的理解。
In Escherichia coli, inconsistencies between in vitro tRNA aminoacylation measurements and in vivo protein synthesis demands were postulated almost 40 years ago, but have proven difficult to confirm. Whole-cell modeling can test whether a cell behaves in a physiologically correct manner when parameterized with in vitro measurements by providing a holistic representation of cellular processes in vivo. Here, a mechanistic model of tRNA aminoacylation, codon-based polypeptide elongation, and N-terminal methionine cleavage was incorporated into a developing whole-cell model of E. coli. Subsequent analysis confirmed the insufficiency of aminoacyl-tRNA synthetase kinetic measurements for cellular proteome maintenance, and estimated aminoacyl-tRNA synthetase kcats that were on average 7.6-fold higher. Simulating cell growth with perturbed kcats demonstrated the global impact of these in vitro measurements on cellular phenotypes. For example, an insufficient kcat for HisRS caused protein synthesis to be less robust to the natural variability in aminoacyl-tRNA synthetase expression in single cells. More surprisingly, insufficient ArgRS activity led to catastrophic impacts on arginine biosynthesis due to underexpressed N-acetylglutamate synthase, where translation depends on repeated CGG codons. Overall, the expanded E. coli model deepens understanding of how translation operates in an in vivo context.
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影响因子：
4
作者：
通讯作者：
--
影响因子：
14.9
作者：
Clifton BE;Fariz MA;Uechi GI;Laurino P
通讯作者：
Laurino P
影响因子：
14.9
作者：
DERRICK, WB;HOROWITZ, J
通讯作者：
HOROWITZ, J
DOI：
10.1126/science.aav3751
发表时间：
2020-07-24
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Macklin DN;Ahn-Horst TA;Choi H;Ruggero NA;Carrera J;Mason JC;Sun G;Agmon E;DeFelice MM;Maayan I;Lane K;Spangler RK;Gillies TE;Paull ML;Akhter S;Bray SR;Weaver DS;Keseler IM;Karp PD;Morrison JH;Covert MW
通讯作者：
Covert MW
影响因子：
14.9
作者：
Keseler IM;Mackie A;Peralta-Gil M;Santos-Zavaleta A;Gama-Castro S;Bonavides-Martínez C;Fulcher C;Huerta AM;Kothari A;Krummenacker M;Latendresse M;Muñiz-Rascado L;Ong Q;Paley S;Schröder I;Shearer AG;Subhraveti P;Travers M;Weerasinghe D;Weiss V;Collado-Vides J;Gunsalus RP;Paulsen I;Karp PD
通讯作者：
Karp PD