Teth137, a Conserved Factor of Unknown Function from Thermoanaerobacter ethanolicus JW200, Represses the Transcription of the adhE Gene In Vitro

Abstract

Teth137, a 13.7 kD protein of unknown function from Thermoanaerobacter ethanolicus JW200, is encoded by 360 nucleotides and has been obtained by DNA-coupled column previously. However, no function study of Teth137 has been published. Homologous modeling of Teth137 shows the protein is comprised of a helix-turn-helix motif which is a typical DNA-binding domain. Therefore, it is speculated Teth137 is a DNA-binding protein and involved in transcription of the adhE gene (encodes alcohol dehydrogenase E). To investigate the function of Teth137, recombinant Teth137 is overexpressed in Escherichia coli JM109 and purified by DEAE column. Purified Teth137 exhibits the affinity with the adhE promoter region in gel electrophoresis mobility shift assay (GEMSA). Teth137 at the concentration of 48 μM retards the migration of 5 nM of probe in the presence of the competitor DNA. Mutant analysis indicates that S69, T70, P71 and T72 are critical to protein–DNA interface; Gly substitutions at these residues results in the loss of the binding ability with the adhE promoter region. Moreover, T. ethanolicus JW200 RNA polymerase, σ subunit and template plasmid are prepared for in vitro transcription assay to detect the regulation function of Teth137. The results of the in vitro transcription show that the transcription of 5 nM of the template plasmid is inhibited by 48 μM of Teth137.