Liang S T, Dennis P P, Bremer H
Molecular and Cell Biology Programs, University of Texas at Dallas, Richardson, Texas 75083-0688, USA.
J Bacteriol. 1998 Dec;180(23):6090-100. doi: 10.1128/JB.180.23.6090-6100.1998.
The expression of lacZ has been analyzed and compared in a series of promoter cloning vectors by measuring the amount of lacZ mRNA by hybridization and the amount of beta-galactosidase by standard enzymatic assay. Expression was driven by the promoter, Pspc, of the spc ribosomal protein operon. The vectors contained either the standard W205 trp-lac fusion with the trp operon transcription terminator, trpt, located in the lacZ leader sequence, or a deletion derivative that functionally inactivates trpt. In the presence of trpt, lacZ expression was temperature dependent so that increasing the growth temperature reduced the accumulation of lacZ mRNA and beta-galactosidase activity. The frequency of transcript termination at trpt was estimated to be near zero at 20 degreesC and at about 45% at 37 degreesC. The amount of Pspc-derived lacZ mRNA and the amount of beta-galactosidase produced per lacZ mRNA varied, depending on the mRNA 5' leader sequence between Pspc and lacZ. These results demonstrate that the quantitative assessment of promoter activities with promoter cloning vectors requires careful analysis and interpretation. One particular construct without trpt did not seem to contain fortuitous transcription or translation signals generated at the fusion junction. In this strain, lacZ expression from Pspc was compared at the enzyme activity and mRNA levels with a previously constructed strain in which lacZ was linked to the tandem P1 and P2 promoters of the rrnB operon. At any given growth rate, the different activities of beta-galactosidase in these two strains were found to reflect the same differences in their amounts of lacZ mRNA. Assuming that the promoter-lacZ fusions in these strains reflect the properties of the promoters in their normal chromosomal setting, it was possible to estimate the absolute transcription activity of Pspc and the relative translation efficiency of Pspc-lacZ mRNA at different growth rates. Transcription from the spc promoter was found to increase from about 10 transcripts per min at a growth rate of 1.0 doublings/h to a maximum plateau of about 23 transcripts per min at growth rates above 1.5 doublings/h. The translation frequency of lacZ mRNA expressed from Pspc was unaffected by growth rates.
通过杂交测量lacZ mRNA的量以及通过标准酶促测定法测量β-半乳糖苷酶的量，对一系列启动子克隆载体中lacZ的表达进行了分析和比较。表达由spc核糖体蛋白操纵子的启动子Pspc驱动。这些载体要么包含标准的W205 trp-lac融合体，其trp操纵子转录终止子trpt位于lacZ前导序列中，要么包含一个在功能上使trpt失活的缺失衍生物。在trpt存在的情况下，lacZ表达是温度依赖性的，因此提高生长温度会降低lacZ mRNA和β-半乳糖苷酶活性的积累。据估计，trpt处转录本终止的频率在20℃时接近零，在37℃时约为45%。源自Pspc的lacZ mRNA的量以及每个lacZ mRNA产生的β-半乳糖苷酶的量各不相同，这取决于Pspc和lacZ之间的mRNA 5'前导序列。这些结果表明，使用启动子克隆载体对启动子活性进行定量评估需要仔细的分析和解释。一种没有trpt的特定构建体似乎不包含在融合连接处产生的偶然转录或翻译信号。在该菌株中，将来自Pspc的lacZ表达在酶活性和mRNA水平上与先前构建的菌株进行了比较，在先前构建的菌株中，lacZ与rrnB操纵子的串联P1和P2启动子相连。在任何给定的生长速率下，发现这两种菌株中β-半乳糖苷酶的不同活性反映了它们lacZ mRNA量的相同差异。假设这些菌株中的启动子-lacZ融合体反映了它们在正常染色体环境中启动子的特性，就有可能估计Pspc的绝对转录活性以及Pspc-lacZ mRNA在不同生长速率下的相对翻译效率。发现从spc启动子的转录从生长速率为1.0代/h时的约每分钟10个转录本增加到生长速率高于1.5代/h时的约每分钟23个转录本的最大平台期。由Pspc表达的lacZ mRNA的翻译频率不受生长速率的影响。
