Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli.
Effects of amino acid starvation on RelA diffusive behavior in live Escherichia coli.
复制标题
氨基酸饥饿对活大肠杆菌中的Rela扩散行为的影响。
DOI：
10.1111/mmi.13252
复制
发表时间：
2016-02
影响因子：
3.6
通讯作者：
Weisshaar JC
中科院分区：
文献类型：
--
作者：
Li W;Bouveret E;Zhang Y;Liu K;Wang JD;Weisshaar JC
在氨基酸饥饿期间，细菌细胞迅速合成核苷酸(p)ppGpp，导致转录谱的大规模重编程，称为严格反应。(p)ppGpp合成酶RelA被A位点含有未带电tRNA的核糖体激活。目前尚不清楚RelA是与核糖体结合还是在细胞质中游离时发生合成。我们研究了三种大肠杆菌菌株，每种菌株表达不同的rela荧光蛋白（RelA-FP）结构：RelA-YFP， RelA-mEos2和RelA-Dendra2。在正常生长条件和氨基酸饥饿条件下进行了单分子定位和跟踪研究。对三种标记方案的研究使我们能够评估RelA FP标记的潜在问题。扩散轨迹和轴向空间分布表明，氨基酸饥饿诱导了所有三种RelA-FP结构与70S核糖体的净结合。这些数据与RelA与70S核糖体结合时合成(p)ppGpp的模型最为一致。我们提出了饥饿时RelA活性的“短跳跃时间”模型。我们的结果与早期的RelA-Dendra2扩散研究相矛盾，该研究推断核糖体外合成(p)ppGpp。造成这种差异的原因尚不清楚。
During amino acid starvation, bacterial cells rapidly synthesize the nucleotides (p)ppGpp, causing a massive re-programming of the transcriptional profile known as the stringent response. The (p)ppGpp synthase RelA is activated by ribosomes harboring an uncharged tRNA at the A site. It is unclear whether synthesis occurs while RelA is bound to the ribosome or free in the cytoplasm. We present a study of three E. coli strains, each expressing a different RelA-fluorescent protein (RelA-FP) construct: RelA-YFP, RelA-mEos2, and RelA-Dendra2. Single-molecule localization and tracking studies were carried out under normal growth conditions and during amino acid starvation. Study of three labeling schemes enabled us to assess potential problems with FP labeling of RelA. The diffusive trajectories and axial spatial distributions indicate that amino acid starvation induces net binding of all three RelA-FP constructs to 70S ribosomes. The data are most consistent with a model in which RelA synthesizes (p)ppGpp while bound to the 70S ribosome. We suggest a “short hopping time” model of RelA activity during starvation. Our results contradict an earlier study of RelA-Dendra2 diffusion that inferred off-ribosome synthesis of (p)ppGpp. The reasons for the discrepancy remain unclear.