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Gene Expression Regulation
The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology.
Lac-repressor DNA interaction in vitro
Transcription factors and DNA-binding proteins bind their specific target sequences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding along non-specific DNA. One important issue in the field of protein-DNA interaction is the understanding of how proteins interact with non-cognate DNA sequences and how they find the sequence of interest along the DNA. We study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers.
The improved resolution of our method allows measurements at physiological salt concentrations, contrary to previous single-molecule in vitro measurements, and reveals important novel aspects of LacI target search mechanism [1]. Remarkably, we measure 1D-diffusion with 20 base-pair resolution and find that sliding of LacI along DNA is sequence-dependent. Moreover, by controlling the structural state of a single LacI molecule through inducer molecules, we demonstrate that only the active (inducer-free) LacI conformation slides on DNA during target search, whereas inactive (inducer-bound) LacI weakly interacts with DNA and does not show sliding. This might be an important regulatory mechanism in vivo to switch off sliding and minimize DNA crowding when the repressor is inactive. Last, the transition from sliding to strong binding to the target sequence requires an additional load-dependent conformational change of LacI. Our data reveal how LacI target search process is regulated by molecular switching and DNA sequence and provide a comprehensive model of LacI facilitated diffusion mechanism
[1] Tempestini, A. et al., Nucleic Acids Research 46, 5001–5011, June 2018
Imaging gene expression in living cells
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