Bauer B F, Elford R M, Holmes W M
Department of Microbiology, Virginia Commonwealth University and Medical College of Virginia, Richmond 23298-0678.
Mol Microbiol. 1993 Jan;7(2):265-73. doi: 10.1111/j.1365-2958.1993.tb01117.x.
The leuV promoter which produces tRNA(1Leu) in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth-rate-dependent regulation (GDR). Each mutant promoter was ligated with a beta-galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the -10 consensus region, when altered, caused remarkable up-promoter effects, increasing efficiency in some cases almost 10-fold. One up mutation which had five successive T residues in the 'discriminator' region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the -35 region to bring it to consensus increased promoter strength 18-fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR-disturbed. All upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.
为了确定序列改变对启动子效率（强度）以及对生长速率依赖性调控（GDR）的影响，已对在大肠杆菌中产生tRNA（1Leu）的leuV启动子进行了广泛的诱变。通过噬菌体λ溶原化将每个突变启动子与β-半乳糖苷酶报告基因连接到宿主细胞的染色体中。在选定的培养基中以不同生长速率生长的细胞，测定报告基因的活性。位于-10共有区域侧翼的序列，当发生改变时，会引起显著的启动子增强效应，在某些情况下效率提高近10倍。在“鉴别区域”有五个连续T残基的一个增强突变完全消除了GDR，而在鉴别区域有单个碱基变化的几个突变对GDR几乎没有影响。另一个在-35区域将一个碱基改变为共有序列的突变使启动子强度增加了18倍，并显著降低了GDR。用his操纵子的片段替换leuV片段的嵌合启动子表明，只有当leuV的鉴别区域被his鉴别区域替换时，GDR才会受到干扰。所有被his序列替换的上游序列对GDR几乎没有影响。总体而言，启动子效率与GDR之间似乎几乎没有相关性。
