TY  - JOUR
AB  - 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 28 bp 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 70-79000 pN/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 -3 s -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.
DA  - 2003
DO  - 10.1016/S1047-8477(03)00127-8
KW  - Sinorhizobium meliloti
KW  - Protein-DNA interaction
KW  - Atomic force microscopy
KW  - Single molecule force spectroscopy
KW  - Galactoglucan
KW  - Regulatory protein
LA  - eng
IS  - 2
M2  - 145
PY  - 2003
SN  - 1047-8477
SP  - 145-152
T2  - Journal of structural biology
TI  - 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
UR  - https://nbn-resolving.org/urn:nbn:de:0070-pub-17731844
Y2  - 2024-11-22T05:14:30
ER  -