Macián F, Pérez-Roger I, Armengod M E
Instituto de Investigaciones Citológicas, Fundación Valenciana de Investigaciones Biomédicas, Valencia, Spain.
Gene. 1994 Jul 22;145(1):17-24. doi: 10.1016/0378-1119(94)90317-4.
We describe a new vector system for the in vitro construction of transcriptional fusions to the lacZ gene, which is expressed from the translational start signals of galK. The galK ribosome-binding site (RBS) and its natural preceding region ensure a constant efficiency for lacZ translation and, thus, the beta-galactosidase (beta Gal) production of a given fusion is directly proportional to the in vivo transcriptional activity of the inserted DNA fragment. Single-copy lambda prophage versions of multicopy constructs can be made by in vivo recombination. We use this system to compare the transcriptional activities of the promoters present in the dnaA-dnaN-recF-gyrB cluster. The order of strength of these promoters is gyrB > dnaA > recF > dnaN. It is assumed that gyrB belongs to the dnaA-dnaN-recF operon, because the short recF-gyrB intercistronic region does not contain a terminator. By using this new vector system, we have detected strong termination signals within recF that are functional even when recF is translated at its normal rate. The low level of transcription coming to the end of recF, and the highest activity of the gyrB promoter, as well as results obtained with several gyrB::lacZ translational fusions, support the conclusion that gyrB is predominantly expressed from its own promoter under standard growth conditions. Finally, we have found that transcription from the dnaA promoters is constant at different growth rates. This supports the idea that autoregulation of the dnaA gene is responsible for the coupling of the DnaA protein synthesis to cell mass increase, and accumulation of DnaA protein governs the initiation of chromosome replication.
我们描述了一种新的载体系统，用于体外构建与lacZ基因的转录融合体，该融合体由galK的翻译起始信号表达。galK核糖体结合位点（RBS）及其天然的前导区域确保了lacZ翻译的恒定效率，因此，给定融合体的β-半乳糖苷酶（βGal）产量与插入DNA片段的体内转录活性直接成正比。多拷贝构建体的单拷贝λ原噬菌体版本可通过体内重组制备。我们使用该系统比较了dnaA-dnaN-recF-gyrB簇中启动子的转录活性。这些启动子的强度顺序为gyrB > dnaA > recF > dnaN。假定gyrB属于dnaA-dnaN-recF操纵子，因为短的recF-gyrB基因间区域不包含终止子。通过使用这种新的载体系统，我们在recF内检测到了强终止信号，即使recF以其正常速率翻译时这些信号也是功能性的。recF末端的低水平转录、gyrB启动子的最高活性以及几种gyrB::lacZ翻译融合体的结果支持了这样的结论，即在标准生长条件下，gyrB主要从其自身启动子表达。最后，我们发现dnaA启动子在不同生长速率下的转录是恒定的。这支持了这样的观点，即dnaA基因的自动调节负责DnaA蛋白合成与细胞质量增加的耦合，并且DnaA蛋白的积累控制染色体复制的起始。
