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Review
. 2021 Jun 30;49(3):1121-1132.
doi: 10.1042/BST20200709.

The needle and the haystack: single molecule tracking to probe the transcription factor search in eukaryotes

Matteo Mazzocca  1 , Tom Fillot  1 , Alessia Loffreda  1 , Daniela Gnani  1 , Davide Mazza  1
Affiliations
Review

The needle and the haystack: single molecule tracking to probe the transcription factor search in eukaryotes

Matteo Mazzocca et al. Biochem Soc Trans. .

Abstract

Transcription factors (TFs) regulate transcription of their target genes by identifying and binding to regulatory regions of the genome among billions of potential non-specific decoy sites, a task that is often presented as a 'needle in the haystack' challenge. The TF search process is now well understood in bacteria, but its characterization in eukaryotes needs to account for the complex organization of the nuclear environment. Here we review how live-cell single molecule tracking is starting to shed light on the TF search mechanism in the eukaryotic cell and we outline the future challenges to tackle in order to understand how nuclear organization modulates the TF search process in physiological and pathological conditions.

Keywords: search mechanism; single molecule; transcription factors.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. Classical search strategies.
(A) Diffusion limited search. Shown is the Smoluchowski relationship for diffusion limited reactions, where τsearch is the time taken by a TF to find a target site, V is the size of the explored volume, a is the target site (typically assumed as the size of a nucleotide, since the TF needs to align to its target with base-pair precision and D is the diffusion coefficient of the molecule). Typical expected values for these paramters are provided in the table. (B) The mechanism of facilitated diffusion speed-up the search process, with an efficiency that depends on the fraction of time the TF spend sliding on DNA.
Figure 2.
Figure 2.. Intranuclear SMT to quantify search times.
(A) in SMT the diffraction limited spots corresponding to individual TF molecules in the nucleus are tracked over time. (B) The single-molecule tracks, that typically last for few frames are segmented into ‘bound’ and ‘free’ molecules and analyzed in terms of the distribution of residence times and fraction of segments belonging to each state. These parameters are then combined to estimate the ‘pseudo-search time τ∗search, that is the time a TF molecule spends on average between two consecutive binding events (at any site).
Figure 3.
Figure 3.. Exploration strategies in the eukaryotic cell nucleus.
The TF exploration strategy can be determined by two numbers, the walk dimension dw and the diffusible space dimension df. (A) The MSD analysis of the diffusing molecules can be used to quantify dw, that is equal to 2 for Brownian diffusion and larger than 2 for hindered (anomalous) diffusion. (B) df describes the space that can be explored by the TF, and it is equal to 1, for molecules sliding on DNA, 2 for molecules exploring surfaces, 3 for molecules diffusing freely in 3D. If the diffusible space is obstructed on multiple scales, df can be smaller than 3. (C) When dw > df, the exploration is compact and the TF visits all the sites of a region of space before leaving it. Oppositely, when dw > df the exploration is non-compact and the TF samples the potential targets only sparsely. Compact and non-compact exploration can be combined together intermittently, and this combination can guide the TF molecules to their target sites speeding up the search process. (D) The anisotropy of the diffusion process (measured as the distribution of angles between consecutive displacements) can inform on the search strategy, as compact exploration should result in high backward anisotropy. (E) Measuring the diffusional anisotropy as function of the distance jumped by the molecules can reveal exploration modes, including the presence of trapping zones that underlie guided exploration.
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