A NUSG-LIKE PROTEIN FROM THERMOTOGA-MARITIMA BINDS TO DNA AND RNA

The NusG-like protein from Thermotoga maritima was expressed in Escher ichia coli and purified to homogeneity, Purified T. maritima NusG exhi bited a generalized, non sequence-specific and highly cooperative DNA and RNA binding activity, The complexes formed between nucleic acid an d T. maritima NusG were unable to penetrate a polyacrylamide or agaros e gel, The affinity of the protein for DNA was highest in buffers cont aining about 50 mM salt. The DNA-protein complexes could not be staine d with ethidium bromide, were resistant to digestion by TaqI endonucle ase, were able to be transcribed in vitro by T. maritima RNA polymeras e, and contained a minimum of about 30 to 40 monomers of NusG per kb o f 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 struct ure that resembles the complex between DNA and histone-like protein Hc l from Chlamydia trachomatis. Neither the wild-type T. maritima nusG g ene nor a deletion derivative more similar to the E. coli gene was abl e to substitute for the essential E. coli nusG. Two variants of the Nu sG protein were constructed, expressed, and purified: one contains onl y the entire 171-amino-acid insertion that is unique to T. maritima Nu sG, 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 1 0-fold lower than that of the wild-type T. maritima NusG.