Abstract
The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. We built a Markov State Model from extensive molecular dynamics simulations to identify metastable intermediate states and the dynamics of backtracking at atomistic detail. Our results reveal that Pol II backtracking occurs in a stepwise mode where two intermediate states are involved. We find that the continuous bending motion of the Bridge helix (BH) serves as a critical checkpoint, using the highly conserved BH residue T831 as a sensing probe for the 3′-terminal base paring of RNA:DNA hybrid. If the base pair is mismatched, BH bending can promote the RNA 3′-end nucleotide into a frayed state that further leads to the backtracked state. These computational observations are validated by site-directed mutagenesis and transcript cleavage assays, and provide insights into the key factors that regulate the preferences of the backward translocation.
Highlights
The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood
The transcription proofreading is enabled by Pol II backtracking: Pol II moves in a reverse direction from the pre-translocation state to a ‘backtracked’ state where the RNA 30-end nucleotide dislodges from the active site, and extrudes through the pore region of the secondary channel[7,8]
1 or 2 backtracked RNA nucleotides can be removed via an intrinsic activity of Pol II, whereas the cleavage of more than two backtracked RNA nucleotides requires the recruitment of the transcription factor IIS (TFIIS)
Summary
The dynamics of the RNA polymerase II (Pol II) backtracking process is poorly understood. The transcription proofreading is enabled by Pol II backtracking (see Fig. 1a for details): Pol II moves in a reverse direction from the pre-translocation state to a ‘backtracked’ state where the RNA 30-end nucleotide dislodges from the active site, and extrudes through the pore region of the secondary channel[7,8]. This backtracking motion can be greatly favoured by damaged DNA template, mismatched base-pairing or a nucleosomal barrier[9,10,11,12] (see Fig. 1b for details). Does backtracking take place in a stepwise or a concerted mode? (that is, Whether the terminal base pair is disrupted before or co-currently in comparison with pol II reverse translocation?) What is the driving force for the backtracking? How does Pol II modulate the energetics of backtracking when mismatch or DNA damage is present?
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