Rodriguez R L, West R W, Heyneker H L, Bolivar F, Boyer H W
Nucleic Acids Res. 1979 Jul 25;6(10):3267-87. doi: 10.1093/nar/6.10.3267.
By employing a system of RNA polymerase binding and restriction endonuclease digestion, we demonstrate that the region in and around the Hind III site of pBR313 and pBR322 is the promoter for the tetracycline (Tc) resistance gene(s). Furthermore, it is shown that this region was transferred intact from pSC101 during the construction of the latter plasmids. The in vitro insertion of a few base pairs at the Hind III site produces a series of "down" promoter mutations in which the level of in vivo Tc resistance is reduced. Sequence analysis of the various promoter mutations revealed significant base pair rearrangements in the region between -40 and -12 of the promoter. While these base alterations do not appear to affect the firm binding of RNA polymerase, they do affect the ability of mutant promoters to initiate transcription. These observations suggest that the region from -40 to -12, previously designated as the "recognition region", is actually involved in the process of initiation of transcription.
通过采用一种RNA聚合酶结合和限制性内切酶消化系统，我们证明pBR313和pBR322的Hind III位点及其周围区域是四环素（Tc）抗性基因的启动子。此外，还表明该区域在构建后一种质粒的过程中从pSC101完整转移而来。在Hind III位点体外插入几个碱基对会产生一系列“下调”启动子突变，其中体内Tc抗性水平降低。对各种启动子突变的序列分析揭示了启动子-40至-12区域内显著的碱基对重排。虽然这些碱基改变似乎不影响RNA聚合酶的牢固结合，但它们确实影响突变启动子启动转录的能力。这些观察结果表明，先前被指定为“识别区域”的-40至-12区域实际上参与了转录起始过程。
