Liao D, Lurz R, Dobrinski B, Dennis P P
Program in Evolutionary Biology, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
J Bacteriol. 1996 Jul;178(14):4089-98. doi: 10.1128/jb.178.14.4089-4098.1996.
The NusG-like protein from Thermotoga maritima was expressed in Escherichia coli and purified to homogeneity. Purified T. maritima NusG exhibited a generalized, non-sequence-specific and highly cooperative DNA and RNA binding activity. The complexes formed between nucleic acid and T. maritima NusG were unable to penetrate a polyacrylamide or agarose gel. The affinity of the protein for DNA was highest in buffers containing about 50 mM salt. The DNA-protein complexes could not be stained with ethidium bromide, were resistant to digestion by TaqI endonuclease, were able to be transcribed in vitro by T. maritima RNA polymerase, and contained a minimum of about 30 to 40 monomers of NusG per kb of duplex DNA. The protein had comparable affinities for duplex DNA and RNA but a lower affinity for single-stranded DNA. Electron microscopy showed that the DNA in the complex is condensed within a large structure that resembles the complex between DNA and histone-like protein Hcl from Chlamydia trachomatis. Neither the wild-type T. maritima nusG gene nor a deletion derivative more similar to the E. coli gene was able to substitute for the essential E. coli nusG. Two variants of the NusG protein were constructed, expressed, and purified: one contains only the entire 171-amino-acid insertion that is unique to T. maritima NusG, and the other has only the sequences present in NusG homologs from E. coli and other eubacteria. Both variants exhibited similar DNA and RNA binding behavior, although their apparent affinities were 5- to 10-fold lower than that of the wild-type T. maritima NusG.
来自嗜热栖热菌的类NusG蛋白在大肠杆菌中表达并纯化至同质。纯化后的嗜热栖热菌类NusG蛋白表现出广泛的、非序列特异性且高度协同的DNA和RNA结合活性。核酸与嗜热栖热菌类NusG形成的复合物无法穿透聚丙烯酰胺或琼脂糖凝胶。该蛋白对DNA的亲和力在含有约50 mM盐的缓冲液中最高。DNA - 蛋白质复合物不能用溴化乙锭染色，对TaqI核酸内切酶的消化具有抗性，能够在体外由嗜热栖热菌RNA聚合酶转录，并且每kb双链DNA至少含有约30至40个NusG单体。该蛋白对双链DNA和RNA具有相当的亲和力，但对单链DNA的亲和力较低。电子显微镜显示，复合物中的DNA在一个大结构中浓缩，该结构类似于沙眼衣原体DNA与类组蛋白Hcl之间的复合物。嗜热栖热菌野生型nusG基因及其与大肠杆菌基因更相似的缺失衍生物均不能替代必需的大肠杆菌nusG。构建、表达并纯化了NusG蛋白的两个变体：一个仅包含嗜热栖热菌类NusG特有的完整171个氨基酸的插入序列，另一个仅具有来自大肠杆菌和其他真细菌的NusG同源物中存在的序列。尽管它们的表观亲和力比野生型嗜热栖热菌类NusG低5至10倍，但两个变体均表现出相似的DNA和RNA结合行为。
