ABSTRACT

DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.

摘要

DnaA 是染色体复制的起始蛋白，但其在肠杆菌中的稳态调节尚不清楚。DnaA 水平在不同的生长速率下保持稳定，表明新陈代谢和dnaA表达之间存在联系。在我们为解开肠杆菌科中 sRNA rnTrpL 的目标而进行的生物信息学预测中，dnaA mRNA 是最保守的目标候选者。sRNA rnTrpL 来源于色氨酸生物合成操纵子的转录衰减子。在大肠杆菌中，由于在没有外部色氨酸供应的情况下转录被去抑制，其在最低限度中的水平高于在丰富培养基中的水平。rnTrpL 基因的过表达和缺失分别降低和增加了dnaA mRNA 的水平。rnTrpL 过量产生后dnaA mRNA 水平的降低取决于hfq和rne。验证了体内rnTrpL 和dnaA mRNA之间的碱基配对。在基本培养基中，ΔtrpL突变体中的oriC水平增加，这与预期的 DnaA 过度生产和染色体复制起始的增加一致。与此一致，染色体 rnTrpL 突变消除了与dnaA增加了dnaA mRNA 和oriC水平。此外，在向基本培养基培养物中添加色氨酸后，野生型中的oriC水平增加。因此，rnTrpL 是一种碱基配对 sRNA，它在大肠杆菌中转录后调节dnaA。此外，我们的数据表明 rnTrpL 有助于 DnaA 稳态依赖于营养物质的可用性，这由细胞中的色氨酸水平表示。