Several important biological processes are initiated by the binding
of a protein to a specific site on the DNA. The strategy adopted by a protein,
called transcription factor (TF), for searching its specific binding site on the
DNA has been investigated over several decades. In recent times the effects
obstacles, like DNA-binding proteins, on the search by TF has begun to receive
attention. RNA polymerase (RNAP) motors collectively move along a segment
of the DNA during a genomic process called transcription. This RNAP trac
is bound to affect the diffusive scanning of the same segment of the DNA by
a TF searching for its binding site. Motivated by this phenomenon, here we
develop a kinetic model where a ‘particle’, that represents a TF, searches
for a specific site on a one-dimensional lattice. On the same lattice another
species of particles, each representing a RNAP, hop from left to right exactly
as in a totally asymmetric simple exclusion process (TASEP) which forbids
simultaneous occupation of any site by more than one particle, irrespective of
their identities. Although the TF is allowed to attach to or detach from any
lattice site, the RNAPs can attach only to the first site at the left edge and
detach from only the last site on the right edge of the lattice. We formulate the search as a first-passage process; the time taken to reach the target site
for the first time, starting from a well defined initial state, is the search time.
By approximate analytical calculations and Monte Carlo (MC) computer
simulations, we calculate the mean search time. We show that RNAP traffic
rectifies the diffusive motion of TF to that of a Brownian ratchet, and the
mean time of successful search can be even shorter than that required in the
absence of RNAP traffic. Moreover, we show that there is an optimal rate of
detachment that corresponds to the shortest mean search time.
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Revealing atomic-scale molecular diffusion of a plant-transcription factor WRKY domain protein along DNA
Transcription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here, we constructed a TF model system using the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on DNA by employing all-atom molecular-dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to one strand of DNA with significant energetic bias compared with the other, or nonpreferred strand. The preferential DNA-strand binding becomes most prominent in the static process, from nonspecific to specific DNA binding, but less distinct during diffusive movements of the domain protein on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base-pair stepping cycle of the protein tracking along major groove of DNA with a homogeneous poly-adenosine sequence, as individual hydrogen bonds break and reform at the protein–DNA binding interface. Further DNA-groove tracking motions of the protein forward or backward, with occasional sliding as well as strand crossing to minor groove of DNA, were also captured. The processive diffusion of WRKY along DNA has been further sampled via coarse-grained MD simulations. The study thus provides structural dynamics details on diffusion of a small TF domain protein, suggests how the protein approaches a specific recognition site on DNA, and supports further high-precision experimental detection. The stochastic movements revealed in the TF diffusion also provide general clues about how other protein walkers step and slide along DNA.
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- Award ID(s):
- 1763272
- NSF-PAR ID:
- 10322252
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 23
- ISSN:
- 0027-8424
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Development of the malaria parasite, Plasmodium falciparum, is regulated by a limited number of sequence-specific transcription factors (TFs). However, the mechanisms by which these TFs recognize genome-wide binding sites is largely unknown. To address TF specificity, we investigated the binding of two TF subsets that either bind CACACA or GTGCAC DNA sequence motifs and further characterized two additional ApiAP2 TFs, PfAP2-G and PfAP2-EXP, which bind unique DNA motifs (GTAC and TGCATGCA). We also interrogated the impact of DNA sequence and chromatin context on P. falciparum TF binding by integrating high-throughput in vitro and in vivo binding assays, DNA shape predictions, epigenetic post-translational modifications, and chromatin accessibility. We found that DNA sequence context minimally impacts binding site selection for paralogous CACACA-binding TFs, while chromatin accessibility, epigenetic patterns, co-factor recruitment, and dimerization correlate with differential binding. In contrast, GTGCAC-binding TFs prefer different DNA sequence context in addition to chromatin dynamics. Finally, we determined that TFs that preferentially bind divergent DNA motifs may bind overlapping genomic regions due to low-affinity binding to other sequence motifs. Our results demonstrate that TF binding site selection relies on a combination of DNA sequence and chromatin features, thereby contributing to the complexity of P. falciparum gene regulatory mechanisms.
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Abstract TMEM16F is a Ca2+-activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca2+ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.
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null (Ed.)Abstract Single-molecule tracking (SMT) allows the study of transcription factor (TF) dynamics in the nucleus, giving important information regarding the diffusion and binding behavior of these proteins in the nuclear environment. Dwell time distributions obtained by SMT for most TFs appear to follow bi-exponential behavior. This has been ascribed to two discrete populations of TFs—one non-specifically bound to chromatin and another specifically bound to target sites, as implied by decades of biochemical studies. However, emerging studies suggest alternate models for dwell-time distributions, indicating the existence of more than two populations of TFs (multi-exponential distribution), or even the absence of discrete states altogether (power-law distribution). Here, we present an analytical pipeline to evaluate which model best explains SMT data. We find that a broad spectrum of TFs (including glucocorticoid receptor, oestrogen receptor, FOXA1, CTCF) follow a power-law distribution of dwell-times, blurring the temporal line between non-specific and specific binding, suggesting that productive binding may involve longer binding events than previously believed. From these observations, we propose a continuum of affinities model to explain TF dynamics, that is consistent with complex interactions of TFs with multiple nuclear domains as well as binding and searching on the chromatin template.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2102621118
