Genetic engineering and characterization of LysR-type transcriptional regulators

Abstract

This thesis describes research aimed at understanding the structure and function of LysR-type transcriptional regulators. I studied two LysR-type proteins. One from the archaeon Methanococcus jannaschii, MJ-LysR. The other is from Burkholderia cepacia, DgdR. The MJ-LysR is the first putative LysR-type transcriptional regulator found in archaea. It is surprising that a prokaryotic transcriptional regulator is present in archaea, whose basal transcription machinery and RNA polymerase are more closely related to those of eukaryotes. To elucidate the structure and function of M-LysR protein, the gene was subcloned and expressed in E. coli. The gene product was isolated and purified by heat treatment and size exclusion chromatography. An in vitro binding assay showed that the purified protein bound to the intergenic region between the lysR gene and its upstream gene specifically and selectively. The results also showed that the protein maintained its binding activity even at 94C̊. The DNA footprinting data demonstrated a 30 bp protected region. Thus, this protein probably regulates expression of its own structural gene and perhaps the adjacent upstream gene. DgdR protein from Burkholderia cepacia had been previously characterized. The previous study showed that 2-methylalanine, the inducer for the DgdR regulated dgdA gene expression, but not D or L-alanine induced the conformational changes on DNA-protein complex. To further confirm this result, eleven amino acids with structures similar to 2-methylalainine were tested for their ability on affecting the binding of the DgdR protein to its operator site. Among these amino acids tested, only 2-methylalanine, 1-aminocyclopentane-1-carboxylic acid, S-2-aminobutanoic acid, RS-isovaline, and 2-trifluoromethyl-2-aminobutanoic acid generated the measurable band shifting. D- or L-norvaline, 2,2-diethyl glycine, and 2-trifluoromethylalanine did not cause any measurable change. It was concluded that both alkyl side chain size and hydrophobicity are important for the inducer recognition and binding in this protein. To solve the problem in DgdR protein purification caused by low solubility of this protein, a dgdR fusion gene to malE gene was constructed. This fusion gene provides a useful tool to further study and crystallize the DgdR protein.