Our results argue against facilitated diffusion at either the microscopic or submicroscopic scales as a significant contributing component of the E. coli RNAP promoter search, and we also show that in general any potential contributions of facilitated diffusion can be overcome through increased protein abundance, even for proteins that can slide long distances on DNA. Facilitated diffusion and 3D collisions can be conceptually considered as two distinct, competing pathways either of which has the potential to result in target binding, and 3D diffusion will always be favored at protein concentrations equal to or exceeding the facilitation threshold simply because the relative increase in protein abundance increases the probability of a direct collision with the target site (). In other words, just because a protein is physically capable of hopping and/or sliding over long distances along DNA does not mean that these processes will accelerate target binding because protein concentration can always have a dominating effect on the overall search process. A broader implication of this conclusion is that proteins present at low concentrations in living cells (e.g. lac repressor, <10 molecules cell−1) may be more apt to locate targets through facilitated diffusion, whereas those present at higher concentrations (e.g. RNAP, ~2,000–3,000 molecules cell−1) may be more likely to engage their target sites through 3D diffusion.
Increasingly complex environments encountered during in vivo searches
Our experimental setting differs substantially from much more complex physiological environments where the promoter search might be influenced by the presence of factors that can assist in the recruitment of RNAP to promoters, or by local DNA folding, higher–order chromatin architecture, and macromolecular crowding (). While we cannot yet quantitatively assess the influence of these parameters, we can consider how they might qualitatively affect the promoter search.
Transcriptional activators, such as catabolite activator protein (CAP), are commonly involved in the regulation of gene expression, and can exert their effects either by facilitating recruitment of RNAP or by stimulating steps after recruitment (e.g.
open complex formation, promoter escape, etc.
In scenarios involving factor–assisted recruitment, additional protein–protein contacts stabilize interactions between RNAP and the promoter. However, the presence of a transcriptional activator near a promoter should not fundamentally alter the search process by causing RNAP to start sliding and/or hopping along the DNA while executing the search, rather it would just make the target appear “larger” to RNAP (i.e.
promoter plus factor, instead of just the promoter), which would in turn reduce the facilitation threshold. Factors that stimulate steps after recruitment would not influence the search because they exert their effects only after the promoter search is complete.
Higher–order organization of DNA in vivo
has the potential to promote 3D collisions or “jumps”, but is not expected to favor 1D sliding and/or hopping, both of which can be considered as local events that are not influenced by global DNA architecture.18,20
In contrast, naked DNA stretched out at low dilution presents the most favorable possible conditions for 1D sliding and/or hopping.17,21
The fact that we do not detect facilitated diffusion contributing to the promoter search by RNAP under conditions that should otherwise greatly favor hopping and/or sliding suggests these processes are unlikely to occur in vivo
simply due to the more complex 3D DNA environment.
Molecular crowding, either in solution or on the DNA, is a nontrivial issue, which can have both positive and negative impacts on DNA binding. Increased nonspecific binding can arise from macromolecular crowding in solution due to excluded volume effects,53
and any increase in nonspecific binding has the potential to promote facilitated diffusion. Although in the case of E. coli
RNAP, increased nonspecific binding brought about through use of low ionic strength conditions still does not lead to microscopically detectable 1D diffusion, suggesting any increased nonspecific affinity caused by excluded volume effects is unlikely to cause RNAP to start diffusing along DNA. The effects of macromolecular crowding on DNA arise from the presence of other nonspecific DNA–binding proteins, which can reduce nonspecific DNA–binding affinities through competitive inhibition,54
and can also impede 1D diffusion along DNA through steric hindrance.18,19,36
The net result of the seemingly opposed influences of macromolecular crowding in solution versus molecular crowding on the DNA has yet to be quantitatively explored, although one might anticipate that highly abundant proteins such as Fis and HU (each of which can be present at concentrations of up to ~30–50 μM in E. coli
) would disfavor facilitated searches by restricting access to nonspecific sites.19
In summary, there are at least four reasons why promoter searches in E. coli
would not benefit from facilitated diffusion. First, there are on the order of ~2,000–3,000 molecules of RNAP in E. coli
, corresponding to an in vivo
concentration of ~2–3 μM.55
Based on our findings, if even a small fraction of the total RNAP present in a cell were free, then it should still locate promoters through 3D collisions rather than facilitated diffusion. Estimates have suggested that there are on the order of ~550 molecules (~0.5 μM) of free σ70
–containing RNAP holoenzyme in living bacteria;55
if these estimates are correct, then the facilitation threshold would have to somehow increase by roughly three orders of magnitude in order for hopping and or sliding to accelerate the promoter search in vivo
. In contrast to RNAP, lac repressor, which is thought to employ facilitated diffusion in vivo
during its target search,45,56
may need to do so to compensate for its much lower intracellular abundance (<10 molecules cell−1
) and the corresponding scarcity of its targets (3 lac operators per genome). Second, long nonspecific lifetimes will lead to slower searches, so RNAP appears to be optimized to avoid wasting time scanning nonspecific DNA.10-13,17
Third, other proteins (e.g.
Fis, HU, IHF, H–NS, etc.
) may obstruct 1D–diffusion, but such obstacles could be avoided through 3D–searches.19
Fourth, other steps are rate–limiting during gene expression (e.g.
promoter accessibility, promoter escape, elongation, etc.
suggesting there is simply no need for RNAP to locate promoters faster than the 3D–diffusion limit. Finally, despite the much more complicated environments present in physiological settings, our general conclusion regarding the effects of protein abundance on target searches should remain qualitatively true because higher protein concentrations will increase the probability of direct target binding through 3D collisions.