Dynamics of chromosomal target search by a membrane-integrated one-component receptor

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.

A nonstop thrill ride from genes to the assembly of the T3SS injectisome

“…The very first step of the induction process—capturing the vtrB locus of Vp-PAI by VtrAC—looks like searching for a needle in a haystack. However, the mission is possible based on what is known about other systems: e.g., it takes just about 10 min for the transmembrane pH receptor CadC to bind its distinctive target site on the chromosome of E. coli 32 . The membrane location of VtrB in the immediate vicinity of its locus is also not obvious: while nascent proteins are expected to be linked to the gene through transertion, the final VtrB cluster is presumably so large that its diffusion in the membrane may be severely hampered.…”

Modeling Receptor Motility along Advecting Lipid Membranes

“…Martini et al [ 75 ] studied the kinetics of a membrane-integrated protein that locates at specific binding sites on the genome, and also acts as a transcriptional activator. Mathematical analysis and KMC simulations of lattice models were combined with fluorescence-microscopy experiments.…”