Abstract
Membrane proteins account for about one third of the cellular proteome, but it is still unclear how dynamic they are and how they establish functional contacts with cytoplasmic interaction partners. Here, we consider a membrane-integrated one-component receptor that also acts as a transcriptional activator, and analyze how it kinetically locates its specific binding site on the genome. We focus on the case of CadC, the pH receptor of the acid stress response Cad system in E. coli. CadC is a prime example of a one-component signaling protein that directly binds to its cognate target site on the chromosome to regulate transcription. We combined fluorescence microscopy experiments, mathematical analysis, and kinetic Monte Carlo simulations to probe this target search process. Using fluorescently labeled CadC, we measured the time from activation of the receptor until successful binding to the DNA in single cells, exploiting that stable receptor-DNA complexes are visible as fluorescent spots. Our experimental data indicate that CadC is highly mobile in the membrane and finds its target by a 2D diffusion and capture mechanism. DNA mobility is constrained due to the overall chromosome organization, but a labeled DNA locus in the vicinity of the target site appears sufficiently mobile to randomly come close to the membrane. Relocation of the DNA target site to a distant position on the chromosome had almost no effect on the mean search time, which was between four and five minutes in either case. However, a mutant strain with two binding sites displayed a mean search time that was reduced by about a factor of two. This behavior is consistent with simulations of a coarse-grained lattice model for the coupled dynamics of DNA within a cell volume and proteins on its surface. The model also rationalizes the experimentally determined distribution of search times. Overall our findings reveal that DNA target search does not present a much bigger kinetic challenge for membrane-integrated proteins than for cytoplasmic proteins. More generally, diffusion and capture mechanisms may be sufficient for bacterial membrane proteins to establish functional contacts with cytoplasmic targets.
Highlights
Bacteria are exposed to fluctuating environments with frequent changes in nutrient conditions and communication signals, and life-threatening conditions such as environmental stresses and antibiotics [1]
Adaptation to changing environments is vital to bacteria and is enabled by sophisticated signal transduction systems
How can a membrane-integrated protein bind to specific sites on the genome to regulate transcription? Here, we study the kinetics of this process, which involves both protein diffusion within the membrane and conformational fluctuations of the genomic DNA
Summary
Bacteria are exposed to fluctuating environments with frequent changes in nutrient conditions and communication signals, and life-threatening conditions such as environmental stresses and antibiotics [1]. The target search dynamics of cytoplasmic transcription factors have been thoroughly studied in the past decades, triggered by early in vitro experiments indicating that the Escherichia coli Lac repressor finds its target site faster than the rate limit for three-dimensional (3D) diffusion [4]. Compared to pure 3D diffusion, sliding increases the association rate by effectively enlarging the target size (“antenna” effect) [7,8,9]. These dynamics were later probed with single-molecule methods, both in vitro [10, 11] and in vivo [12, 13]. Further studies continued to add to the detailed understanding of this target search process, e.g. with respect to effects of DNA conformation [14], DNA dynamics [15], and macromolecular crowding [16]
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