Nozaki N, Okazaki T, Ogawa T
Department of Molecular Biology, School of Science, Nagoya University, Japan.
J Biol Chem. 1988 Oct 5;263(28):14176-83.
Transcription was carried out in vitro by RNA polymerase on supercoiled minichromosome DNA containing the replication origin of the Escherichia coli chromosome (oriC) and the products were analyzed. Leftward transcription starting from the 16-kDa gene promoter located adjacent to the right of oriC was inhibited by the DnaA protein, a protein essential for initiation of replication. This inhibition is due to binding of DnaA protein to the 9-base pair sequence (dnaA box) located just upstream of the 16-kDa gene promoter. The inhibition was observed at levels of DnaA protein and RNA polymerase comparable to those required for replication of oriC plasmids in vitro and was abolished at high levels of RNA polymerase. These results suggest that a balance of DnaA protein and RNA polymerase is important in regulating transcription and that this regulation operates under conditions that allow initiation of replication. The transcription traversed oriC with very few transcripts terminating before or within it. DnaA protein did not affect the chain length of these transcripts. Furthermore, we identified leftward transcripts which start close to position 177 in the oriC sequence.
转录在体外由RNA聚合酶在含有大肠杆菌染色体复制起点（oriC）的超螺旋微型染色体DNA上进行，并对产物进行分析。从位于oriC右侧相邻的16 kDa基因启动子开始的向左转录受到DnaA蛋白的抑制，DnaA蛋白是复制起始所必需的一种蛋白质。这种抑制是由于DnaA蛋白与位于16 kDa基因启动子上游的9碱基对序列（dnaA框）结合所致。在与体外oriC质粒复制所需水平相当的DnaA蛋白和RNA聚合酶水平下观察到了这种抑制，并且在高水平的RNA聚合酶下这种抑制被消除。这些结果表明，DnaA蛋白和RNA聚合酶的平衡在调节转录中很重要，并且这种调节在允许复制起始的条件下发挥作用。转录穿过oriC，在oriC之前或其中终止的转录本很少。DnaA蛋白不影响这些转录本的链长。此外，我们鉴定出了在oriC序列中靠近位置177处开始的向左转录本。
