Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins.
The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.
Department of Botany-Microbiology, University of Oklahoma, Norman 73019, USA.
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D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010582:Bacteriophage lambda / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000235:genetics*; D008969:Molecular Sequence Data; D010445:Peptide Elongation Factors; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012685:Sepharose; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014158:Transcription, Genetic; D035602:Transcriptional Elongation Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; C068727:nusA protein, E coli; C087294:Q protein, Bacteria; D012685:Sepharose; D005982:Glutathione Transferase; D012321:DNA-Directed RNA Polymerases; D013917:Thrombin