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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.

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 of the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on the DNA employing all-atom molecular dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to the Crick strand of DNA with significantly stronger energetic association than to the Watson strand. The preferential binding becomes highly prominent from non-specific to specific DNA binding, but less distinct from static binding to diffusive movements of WRKY on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base pair (bp) stepping cycle of WRKY tracking along major groove of DNA with homogenous (AT) n sequence, as individual protein-DNA contacts break and reform at the binding interface. Continuous tracking of WRKY forward or backward, with occasional sliding as well as strand crossing to the minor groove of DNA, have also been captured in the simulation. The processive diffusion of WRKY had been confirmed by accompanied single-molecule fluorescence assays and coarse-grained (CG) structural simulations. The study thus provides unprecedented structural dynamics details on the TF diffusion, suggests how TF possibly approaches to gene target, and supports further high-precision experimental follow-up. The stochastic movements revealed in the TF diffusion also provide general clues on how other nucleic acid walkers step and slide along DNA. Significance Statement How transcription factors search for target genes impact on how quickly and accurately the genes are transcribed and expressed. To locate target sufficiently fast, 1D diffusion of the protein along DNA appears essential. Experimentally, it remains challenging to determine diffusional steps of protein on DNA. Here, we report all-atom equilibrium simulations of a WRKY protein binding and diffusing on DNA, revealing structural dynamics details which have not been identified previously. We unprecedently demonstrate a complete stepping cycle of the protein for one base pair on DNA within microseconds, along with stochastic stepping or sliding, directional switching, and strand crossing. Additionally, we have found preferential DNA strand association of WRKY. These suggest how protein factors approach toward target DNA sequences.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA-DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.