Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins

Abstract

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Towards identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions, rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid does not pose a physical barrier for protein diffusion, but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58-99%) of their search time bound to DNA and occupy as much as ~30% of the chromosomal DNA at any time. Chromosome-crowding likely has important implications for the function of all DNA-binding proteins.

Single-molecule tracking reveals the functional allocation,in vivointeractions and spatial organization of universal transcription factor NusG

“…2). Given that NusG is not known to bind directly to chromosomal DNA, but instead binds to the chromosome indirectly (via RNAP during transcription elongation), we would expect that the D* value and diffusion behaviour of NusG should be independent of the presence of the chromosome, as we have shown for proteins lacking DNAbinding domains (Stracy et al, 2021). However, the D* value for free NusG matches that for a 4times larger protein that is unable to bind DNA (Lac -41 , a ~200 KDa truncated lac repressor (LacI) derivative lacking its DNA-binding domain).…”

“…Further, under the same consideration that NusG does not bind directly to chromosomal DNA, the diffusion of free NusG in cells with intact nucleoids should resemble the behaviour of HU-PAmCherry, 48 KDa (same size as NusG-PAmCherry) in cells where the chromosomal DNA has been degraded (DNA-free cells; see (Stracy et al, 2021)). Again, strikingly, while the estimated accurate diffusion coefficient of HU-PAmCherry in DNA-free cells is Dacc ~12.6 μm 2 /s (Stracy et al, 2021), the Dacc for NusG-PAmCherry in cells with intact nucleoids is estimated to be ~3.5 μm 2 /s (based on the similarity of the D* values for NusG-PAmCherry and LacI-PAmCherry, and the conversion of D* to Dacc for LacI-PAmCherry using simulations of diffusion (Stracy et al, 2021)).…”

“…Here, we advance our understanding of NusG functions by examining how NusG distributes between its various activities in vivo. To achieve this, we performed single-molecule tracking and super-resolution imaging of NusG fusions with photoactivatable protein PAmCherry, an approach we previously used to study proteins involved in transcription, DNA repair, and chromosome organization (Garza de Leon et al, 2017;Stracy et al, 2015Stracy et al, , 2021Uphoff et al, 2013;Zawadzki et al, 2015). Under conditions supporting moderate growth rates, we found that most of NusG is present in a chromosome-associated population (via an elongating RNAP) and a slowly-diffusing population corresponding to a NusG complex with free 30S ribosomal subunit; notably, there is little free NusG in the cell.…”

Role of Salt Valency in the Switch of H-NS Proteins between DNA-Bridging and DNA-Stiffening Modes

“…This latter description emerges quite naturally when looking at atomic force micrographs of DNA/protein complexes deposited on mica plates ( 5), but simulations have shown that the conformations of DNA/protein complexes are significantly different in bulk and in planar conditions and that the parallel protein bridges geometry arises from the rearrangement of the complexes after their deposition on the charged surface (38,39). Moreover, recent single-molecule experiments have reported the presence of clusters of H-NS proteins buried inside the nucleoid (46), while other experiments have shown that mutants of H-NS, which have disrupted dimer-dimer interactions and cannot form higher order oligomers, are also unable to form bridges between two DNA duplexes (47). The ability for H-NS dimers to clusterize therefore appears as a prerequisite for their ability to bridge distant DNA sites, which strongly supports the conclusions of the present work.…”