In agreement with earlier studies (Gogala et al., 2014), the translocating state of Sec61 has no large-scale changes in its architecture.
We report here the crystal structure of RNA polymerase II in the third state, the reverse translocated, or "backtracked" state.
These observations are consistent with a model where NusA shifts the equilibrium of the translocation step toward the pre-translocated state.
Alternatively, it is possible that the system moves back-and-forth spontaneously, driven by thermal energy, along the mechanical coordinate, until a chemical transition, that occurs when the system is in the post-translocated state, prevents the back-translocation and 'rectifies' this random motion into directed motion.
This recognition restricts the oscillation of the EC between translocation states, and if translocation equilibrium is shifted towards the pre-translocated state the recognized sequence may cause a pause of transcription.
If continued translocation is then faster than refolding, the protein will be trapped in an unfolded state, translocated into the degradation chamber, and proteolyzed into small peptides.
We hypothesized that if recognition of the RNA DNA hybrid sequence can stabilize RNAP in the pre-translocated state, it should cause a pause of transcription by restricting translocation into elongation-competent post-translocated state.
RNAP passes through the pre-translocated state at every NAC.
Between the pre-translocated state and the post-translocated state, the mutant is significantly more biased toward the former than the wild type.
Two kinetic models have explicitly calculated the energy differences between the pre-translocated state, the post-translocated state, and backtracked states at each position on DNA.
The enzyme can also enter the pausing pathway by transiting from the pre-translocated state to the backtracked states.
