Solomon K A, Brusilow W S
Department of Chemistry and Biochemistry, University of Maryland, College Park 20742.
J Biol Chem. 1988 Apr 15;263(11):5402-7.
Plasmid pRPG54, which carries the genes for the eight subunits of the proton-translocating ATPase of Escherichia coli, has been found to carry a single base change of a G to an A in the ribosome-binding site for uncE, the gene which codes for the N,N'-dicyclohexylcarbodiimide-binding subunit c of the Fo. This noncoding region mutation both lowers expression of uncE by a factor of 2-3 and affects the function of the ATPase, specifically of the Fo sector. The presence of the mutation results in a decrease in the proton permeability of the Fo or of the entire F1Fo-ATPase complex when either is synthesized from genes on a multicopy plasmid. Expression of uncE from an F1Fo plasmid carrying the wild type ribosome binding site results in increased membrane proton permeability and decreased ability of the resultant ATPase to couple a transmembrane proton gradient to ATP synthesis both in vitro and in vivo. Also, although an Fo plasmid carrying the correct ribosome-binding site causes harmful, F1-dependent proton permeability in unc+ cells (Brusilow, W. S. S. (1987) J. Bacteriol. 169, 4984-4990), an identical plasmid carrying the mutation does not, even though it still codes for a functional reconstitutable Fo. The results show a relationship between the relative level of expression of uncE from a multicopy plasmid and the assembly pathway, proton permeability, and energy-coupling characteristics of the ATPase.
携带大肠杆菌质子转运ATP酶八个亚基基因的质粒pRPG54，已发现在编码Fo的N，N'-二环己基碳二亚胺结合亚基c的uncE基因的核糖体结合位点存在一个从G到A的单碱基变化。这个非编码区突变既使uncE的表达降低2 - 3倍，又影响ATP酶的功能，特别是Fo区段的功能。当从多拷贝质粒上的基因合成Fo或整个F1Fo - ATP酶复合物时，该突变的存在会导致Fo或整个F1Fo - ATP酶复合物的质子通透性降低。从携带野生型核糖体结合位点的F1Fo质粒表达uncE，会导致膜质子通透性增加，并且在体外和体内使所得ATP酶将跨膜质子梯度与ATP合成偶联的能力降低。此外，尽管携带正确核糖体结合位点的Fo质粒在unc +细胞中会导致有害的、F1依赖的质子通透性（Brusilow，W. S. S.（1987）J. Bacteriol. 169，4984 - 4990），但携带该突变的相同质粒却不会，即使它仍然编码功能性可重组的Fo。结果表明了从多拷贝质粒表达uncE的相对水平与ATP酶的组装途径、质子通透性和能量偶联特性之间的关系。
