A NusG-like protein from Thermotoga maritima binds to DNA and RNA
A NusG-like protein from Thermotoga maritima binds to DNA and RNA
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DOI：
10.1128/jb.178.14.4089-4098.1996
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发表时间：
1996-07-01
影响因子：
3.2
通讯作者：
Dennis, PP
中科院分区：
文献类型：
--
作者：
Liao, DQ;Lurz, R;Dennis, PP
在大肠杆菌中表达并纯化了海栖热袍菌NusG样蛋白。maritima NusG具有广泛的、非序列特异性的、高度协同的DNA和RNA结合活性。maritima NusG不能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-蛋白质复合物不能被溴化乙锭染色，能抵抗TaqI内切酶的消化，能在体外被T. maritima RNA聚合酶，并且每kb双链体DNA含有最少约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浓缩在一个大的结构中，类似于DNA和沙眼衣原体的组蛋白样蛋白Hcl之间的复合物。野生型T. maritima nusG基因也不是与E. coli基因能够替代必需的E. coli nusG.构建、表达和纯化了NusG蛋白的两种变体：一种仅包含T. maritima NusG，另一个仅具有来自E.大肠杆菌和其他真细菌。这两种变体表现出相似的DNA和RNA结合行为，尽管它们的表观亲和力比野生型T.海洋营养
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.