Specific binding of the regulatory protein ExpG to promoter regions of the galactoglucan biosynthesis gene cluster of Sinorhizobium meliloti - A combined molecular biology and force spectroscopy investigation

Specific protein-DNA interaction is fundamental for all aspects of gene transcription. We focus on a regulatory DNA-binding protein in the Gram-negative soil bacterium Sinorhizobium meliloti 2011, which is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. The ExpG protein plays a central role in regulation of the biosynthesis of the exopolysaccharide galactoglucan, which promotes the establishment of symbiosis. ExpG is a transcriptional activator of exp gene expression. We investigated the molecular mechanism of binding of ExpG to three associated target sequences in the exp gene cluster with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). Binding of ExpG to expA1, expG-expD1, and expE1 promoter fragments in a sequence specific manner was demonstrated, and a 28bp conserved region was found. AFM force spectroscopy experiments confirmed the specific binding of ExpG to the promoter regions, with unbinding forces ranging from 50 to 165pN in a logarithmic dependence from the loading rates of 70-79000pN/s. Two different regimes of loading rate-dependent behaviour were identified. Thermal off-rates in the range of k_off=(1.2±1.0)×10^-3s^-1 were derived from the lower loading rate regime for all promoter regions. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to the molecular binding mechanism.