Bridge helix bending promotes RNA polymerase II backtracking through a critical and conserved threonine residue.

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

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes.

The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the "secondary channel" is the only route for NTP to reach the active site of the enzyme or if the "main channel" could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription

A two-state mechanism for the PP_i release in RNAP revealed by the MSM.

<p>(A) Two metastable states (S1 and S2) are identified. 500 randomly selected conformations from each metastable state are superimposed and represented with cyan and green spheres for S1 and S2 respectively. Each sphere indicates the coordinate of the center of mass of the PP_i group. (B) The two metastable states are displayed as two circles, and the size of these circles is proportional to the equilibrium populations of the S1 (12.6%±0.02%) and S2 (87.4%±0.02%) state, (C) Key interactions between (Mg-PP_i)²⁻ group and RNAP in each state are displayed. (D) Conservation analysis of the positively charged residues that interact with the (Mg-PP_i)²⁻ group among different species. The sequence alignment was performed using the online software ClustalW2 (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>).</p

Comparison of the secondary channel (in wheat) of RNA Pol II (A) and RNAP (B).

<p>For both structures, RNA, template DNA and non-template DNA are shown in red, cyan and green, respectively. The (Mg-PP_i)²⁻ group is represented in stick and sphere models. Several critical residues in the channel: K752, K619, K620, K518 and K880 in RNA Pol II; R1029, K908, K912, K780 and K1360 in RNAP, are highlighted in blue. The Pol II model used to make this figure was taken from our previous study <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003020#pcbi.1003020-Da1" target="_blank">[25]</a>.</p

Cartoon models of the PP_i release in RNAP (A) and Pol II (B).

<p>Cartoon models of the PP_i release in RNAP (A) and Pol II (B).</p

Single mutant simulations reveal the roles of the critical residues H1242, R1239 and R1029 in the PP_i release.

<p>(A) Potential of mean force (PMF) plot along the distance between the PP_i group and Mg²⁺A. The initial conformations of the mutant simulations are highlighted as black spheres (P1 and P2). (B) The distance between the PP_i group and the P_αatom as a function of the simulation time for WT (left panel), R1239A (middle panel), and R1029A (right panel) simulations initiated from P1. We chose this reaction coordination because this distance can directly measure the relative motions between the terminal RNA nucleotide and the PP_i group before it leaves the active site. (C) The same as (B) except that all the simulations were initiated from P2, and the distance between the PP_i group and the Mg²⁺A was shown. In (A) and (C), the S1 and S2 state are highlighted in blue and light green respectively.</p

Structural Model of RNA Polymerase II Elongation Complex with Complete Transcription Bubble Reveals NTP Entry Routes

<div><p>The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the “secondary channel” is the only route for NTP to reach the active site of the enzyme or if the “main channel” could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription.</p></div

MD relaxation of NTP in the main channel and the secondary channel.

<p>(a-b) Initially, the points constituting the entry routes through the main and secondary channels were divided into 20 clusters. The yellow and orange spheres represent the center of each cluster for the main channel and the secondary channel, respectively. (c-d) A NTP molecule (in licorice presentation) accompanied by a bound magnesium atom (sphere representation) was aligned to each of the cluster centers (shown in (a-b)) by its center of mass (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004354#sec007" target="_blank">Methods</a>). These conformations were used as starting points for relaxation via MD simulations (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004354#sec007" target="_blank">Methods</a>). The template DNA, non-template DNA and mRNA strands are shown in cyan, green and red, respectively, with tube and licorice representations. The cut-view of the protein is shown in grey with a surface representation.</p