Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation
Decreasing transcription elongation rate in Escherichia Coli exposed to amino acid starvation
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DOI：
10.1111/j.1365-2958.1992.tb01393.x
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发表时间：
1992-08
影响因子：
3.6
通讯作者：
U. Vogel;M. Sørensen;S. Pedersen;K. Jensen;M. Kilstrup
中科院分区：
文献类型：
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作者：
U. Vogel;M. Sørensen;S. Pedersen;K. Jensen;M. Kilstrup
大肠杆菌中 lacZ 基因转录所需的时间是在指数生长期间和细菌暴露于部分异亮氨酸饥饿的条件下确定的。为此，在诱导后每隔 10 秒从细胞中提取 RNA，并使用与 lacZ mRNA 的开头或末端互补的探针通过 Northern 杂交进行定量。添加诱导剂和“晚期”探针出现杂交信号之间的时间滞后代表 RNA 聚合酶在 lacZ 基因上的渡越时间，该参数和已知的转录序列长度用于计算 lacZ mRNA 链的生长速率。转录延伸率为c。 43 个核苷酸 s-1 在指数增长期间突然下降至 c。异亮氨酸饥饿开始后，当细胞中积累大量浓度的鸟苷四磷酸 (ppGpp) 时，relA+ 菌株中的 20 个核苷酸 s-1 会发生变化。饥饿条件并不影响 lec 启动子的转录起始，但起始的 lacZ mRNA 链的很大一部分从未完成。对于 rel+ 应变，极性是中等的，因为 c。 25% 的起始 lacZ mRNA 链持续形成全长 mRNA，但对于 relA 菌株来说，极性非常强，以至于无法检测到完整的 lacZ mRNA。当细胞缺乏异亮氨酸时，蛋白链伸长率从未受干扰的生长期的 13 个氨基酸 (aa) s-1 下降到大约 6 个氨基酸 s-1。综合起来，这些结果表明 ppGpp 通过抑制 mRNA 链伸长，在降档期间维持转录和翻译之间的耦合方面发挥着重要作用。讨论了该结果对于严格响应过程中稳定 RNA 合成控制的影响。
The time required for transcription of the lacZ gene in Escherichia coli was determined during exponential growth and under conditions, when the bacterium was exposed to partial isoleucine starvation. To do this, RNA was extracted from the cells at 10 s intervals following induction and quantified by Northern hybridization with probes complementary to either the beginning or the end of the lacZ mRNA. The time lag between inducer addition and the appearance of a hybridization signal at the ‘late’ probe represents the transit time for RNA polymerase on the lacZ gene, and this parameter and the known length of the transcribed sequence were used to calculate the lacZ mRNA chain growth‐rate. The transcription elongation rate was c. 43 nucleotides s‐1 during exponential growth and decreased abruptly to c. 20 nucleotides s‐1 in a relA+ strain after the onset of isoleucine starvation, when massive concentrations of guanosine tetraphosphate (ppGpp) accumulated in the cells. The starvation condition did not affect initiation of transcription at the lec‐promoter, but a substantial fraction of the initiated lacZ mRNA chains was never completed. For the rel+ strain the polarity was moderate, since c. 25% of the initiated lacZ mRNA’ chains were continued into full‐length mRNAs, but for the relA strain the polarity was so strong that no completed lacZ mRNA could be detected. The protein chain elongation rates decreased from 13 amino acids (aa) s‐1 in the unperturbed growth phase to approximately 6 aa s‐1, when the cells starved for isoleucine. In combination, these results suggest that ppGpp plays a major role in maintaining the coupling between transcription and translation during the downshift by inhibiting mRNA chain elongation. The implications of this result for the control of stable RNA synthesis during the stringent response are discussed.