Regensburg 2004 – wissenschaftliches Programm

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SYLS: Life Sciences on the Nanometer Scale - Physics Meets Biology

SYLS 4: Symposium "Life Sciences on the Nanometer Scale - Physics Meets Biology"

SYLS 4.2: Vortrag

Donnerstag, 11. März 2004, 10:00–10:15, H 37

Atomic Force Microscopy and Spectroscopy of Specific Protein-DNA Interaction — •F. Bartels¹, B. Baumgarth², A. Becker², R. Ros¹, and D. Anselmetti¹ — ¹Experimental Biophysics, Faculty of Physics, Bielefeld University — ²Genetics, Faculty of Biology, Bielefeld University

Specific protein-DNA interaction is fundamental for all aspects of gene expression. In the soil bacterium Sinorhizobium meliloti 2011, the protein ExpG controls the biosynthesis of polysaccharide polymers, which promote the bacteriums symbiosis with alfalfa plants for means of fixing molecular nitrogen. We investigated the molecular mechanism of binding of ExpG to three associated DNA target sequences both with standard biochemical methods and single molecule force spectroscopy based on the atomic force microscope (AFM). AFM imaging was used in addition to obtain topographical information regarding the process of binding. We demonstrated binding in a sequence specific manner, with unbinding forces ranging from 50 to 165 pN in a logarithmic dependence from the loading rates of 70 to 79,000 pN/s. Two different regimes of loading rate-dependent behaviour were identified. A thermal off-rate k_off = (1.2 ± 1.0) × 10⁻³ s⁻¹ was derived from the lower loading rate regime for all DNA fragments. In the upper loading rate regime, however, these fragments exhibited distinct differences which are attributed to sequence specific details of the binding mechanism.