Vogel U, Sørensen M, Pedersen S, Jensen K F, Kilstrup M
Institute of Biological Chemistry, University of Copenhagen, Denmark.
Mol Microbiol. 1992 Aug;6(15):2191-200. doi: 10.1111/j.1365-2958.1992.tb01393.x.
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 lac-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 as 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.
测定了大肠杆菌中lacZ基因在指数生长期间以及细菌处于部分异亮氨酸饥饿条件下的转录所需时间。为此，在诱导后每隔10秒从细胞中提取RNA，并通过与lacZ mRNA起始或末端互补的探针进行Northern杂交定量。诱导剂添加与“晚期”探针处杂交信号出现之间的时间间隔代表RNA聚合酶在lacZ基因上的转运时间，利用该参数和已知的转录序列长度来计算lacZ mRNA链的生长速率。在指数生长期间，转录延伸速率约为43个核苷酸每秒，在异亮氨酸饥饿开始后，relA+菌株中的延伸速率突然降至约20个核苷酸每秒，此时细胞中积累了大量的鸟苷四磷酸（ppGpp）。饥饿条件不影响lac启动子处的转录起始，但相当一部分起始的lacZ mRNA链从未完成。对于rel+菌株，极性适中，因为约25%起始的lacZ mRNA链能继续转录为全长mRNA，但对于relA菌株，极性很强，以至于检测不到完整的lacZ mRNA。当细胞缺乏异亮氨酸时，蛋白质链延伸速率从未受干扰的生长阶段的13个氨基酸每秒降至约6个氨基酸每秒。综合这些结果表明，ppGpp通过抑制mRNA链延伸，在生长速率下降期间维持转录和翻译的偶联中起主要作用。本文讨论了这一结果对严格反应期间稳定RNA合成控制的意义。
