Kumar A, Malloch R A, Fujita N, Smillie D A, Ishihama A, Hayward R S
Institute of Cell and Molecular Biology, University of Edinburgh, Scotland, U.K.
J Mol Biol. 1993 Jul 20;232(2):406-18. doi: 10.1006/jmbi.1993.1400.
It is known that Escherichia coli promoters having the major minus 10 consensus sequence TATAAT, but lacking significant resemblance to consensus in the minus 35 region, allow transcriptional initiation in vivo and in vitro if they have an additional TGn motif immediately upstream of minus 10. To determine whether region 4.2 of sigma 70, whose normal role is sequence recognition at minus 35, is unnecessary for initiation of transcription at such "extended minus 10" promoters, we modified the sigma 70 gene so as to generate a carboxy-truncated polypeptide lacking the last 84 amino acids and therefore missing region 4.2. Our results show that both the intact and truncated sigma 70 allow purified RNA polymerase to initiate efficiently and specifically at an extended minus 10 promoter, whereas only the intact sigma 70 permits efficient initiation at normal promoters (defined by minus 35 and minus 10 hexamer sequences).
已知具有主要的 -10 共有序列 TATAAT,但在 -35 区域与共有序列缺乏显著相似性的大肠杆菌启动子,如果在 -10 上游紧邻有一个额外的 TGn 基序,则允许在体内和体外进行转录起始。为了确定 σ70 的 4.2 区域(其正常作用是在 -35 处进行序列识别)对于在这种“扩展的 -10”启动子处的转录起始是否不必要,我们对 σ70 基因进行了修饰,以产生一个羧基末端截短的多肽,该多肽缺少最后 84 个氨基酸,因此缺失 4.2 区域。我们的结果表明,完整的和截短的 σ70 都能使纯化的 RNA 聚合酶在扩展的 -10 启动子处高效且特异性地起始转录,而只有完整的 σ70 能在正常启动子(由 -35 和 -10 六聚体序列定义)处进行高效起始。