A NusG-like protein from Thermotoga maritima binds to DNA and RNA.
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.
Liao
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D
Program in Evolutionary Biology, Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Lurz
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R
Dobrinski
B
B
Dennis
P P
PP
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029968:Escherichia coli Proteins; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000648:ultrastructure; D010445:Peptide Elongation Factors / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D012329:RNA, Bacterial / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; C070291:NusG protein, E coli; D010445:Peptide Elongation Factors; D012329:RNA, Bacterial; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors