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1. A generative model for the behavior of RNA polymerase

   https://doi.org/10.1093/bioinformatics/btw599  

   Azofeifa, Joseph G. ; Dowell, Robin D. ; Berger, ed., Bonnie ( September 2016 , Bioinformatics)

   Transcription by RNA polymerase is a highly dynamic process involving multiple distinct points of regulation. Nascent transcription assays are a relatively new set of high throughput techniques that measure the location of actively engaged RNA polymerase genome wide. Hence, nascent transcription is a rich source of information on the regulation of RNA polymerase activity. To fully dissect this data requires the development of stochastic models that can both deconvolve the stages of polymerase activity and identify significant changes in activity between experiments.

   We present a generative, probabilistic model of RNA polymerase that fully describes loading, initiation, elongation and termination. We fit this model genome wide and profile the enzymatic activity of RNA polymerase across various loci and following experimental perturbation. We observe striking correlation of predicted loading events and regulatory chromatin marks. We provide principled statistics that compute probabilities reminiscent of traveler’s and divergent ratios. We finish with a systematic comparison of RNA Polymerase activity at promoter versus non-promoter associated loci.

   Transcription Fit (Tfit) is a freely available, open source software package written in C/C ++ that requires GNU compilers 4.7.3 or greater. Tfit is available from GitHub (https://github.com/azofeifa/Tfit).

   Supplementary data are available at Bioinformatics online.
2. DNA supercoiling-mediated collective behavior of co-transcribing RNA polymerases

   https://doi.org/10.1093/nar/gkab1252  

   Tripathi, Shubham ; Brahmachari, Sumitabha ; Onuchic, José N. ; Levine, Herbert ( December 2021 , Nucleic Acids Research)

   Multiple RNA polymerases (RNAPs) transcribing a gene have been known to exhibit collective group behavior, causing the transcription elongation rate to increase with the rate of transcription initiation. Such behavior has long been believed to be driven by a physical interaction or ‘push’ between closely spaced RNAPs. However, recent studies have posited that RNAPs separated by longer distances may cooperate by modifying the DNA segment under transcription. Here, we present a theoretical model incorporating the mechanical coupling between RNAP translocation and the DNA torsional response. Using stochastic simulations, we demonstrate DNA supercoiling-mediated long-range cooperation between co-transcribing RNAPs. We find that inhibiting transcription initiation can slow down the already recruited RNAPs, in agreement with recent experimental observations, and predict that the average transcription elongation rate varies non-monotonically with the rate of transcription initiation. We further show that while RNAPs transcribing neighboring genes oriented in tandem can cooperate, those transcribing genes in divergent or convergent orientations can act antagonistically, and that such behavior holds over a large range of intergenic separations. Our model makes testable predictions, revealing how the mechanical interplay between RNAPs and the DNA they transcribe can govern transcriptional dynamics.
3. Beyond the canonical role of TFIIB in eukaryotic transcription

   O’Brien M, and Ansari ( April 2022 , Current genetics)

   The role of general transcription factor TFIIB in transcription extends well beyond its evolutionarily conserved function in initiation. Chromatin localization studies demonstrating binding of TFIIB to both the 5’ and 3’ ends of genes in a diverse set of eukaryotes strongly suggested a rather unexpected role of the factor in termination. TFIIB indeed plays a role in termination of transcription. TFIIB occupancy of the 3’ end is possibly due to its interaction with the termination factors residing there. Interaction of the promoter-bound TFIIB with factors occupying the 3’ end of a gene may be the basis of transcription-dependent gene looping. The proximity of the terminator-bound factors with the promoter in a gene loop has the potential to terminate promoter-initiated upstream anti-sense transcription thereby conferring promoter directionality. TFIIB, therefore, is emerging as a factor with pleiotropic roles in the transcription cycle. This could be the reason for preferential targeting of TFIIB by viruses. Further studies are needed to understand the critical role of TFIIB in viral pathogenesis in the context of its newly identified roles in termination, gene looping and promoter directionality.
4. Transcription factor regulation of RNA polymerase’s torque generation capacity

   https://doi.org/10.1073/pnas.1807031116  

   Ma, Jie ; Tan, Chuang ; Gao, Xiang ; Fulbright, Jr., Robert M. ; Roberts, Jeffrey W. ; Wang, Michelle D. ( February 2019 , Proceedings of the National Academy of Sciences)

   During transcription, RNA polymerase (RNAP) supercoils DNA as it translocates. The resulting torsional stress in DNA can accumulate and, in the absence of regulatory mechanisms, becomes a barrier to RNAP elongation, causing RNAP stalling, backtracking, and transcriptional arrest. Here we investigate whether and how a transcription factor may regulate both torque-inducedEscherichia coliRNAP stalling and the torque generation capacity of RNAP. Using a unique real-time angular optical trapping assay, we found that RNAP working against a resisting torque was highly prone to extensive backtracking. We then investigated transcription in the presence of GreB, a transcription factor known to rescue RNAP from the backtracked state. We found that GreB greatly suppressed RNAP backtracking and remarkably increased the torque that RNAP was able to generate by 65%, from 11.2 pN⋅nm to 18.5 pN·nm. Variance analysis of the real-time positional trajectories of RNAP after a stall revealed the kinetic parameters of backtracking and GreB rescue. These results demonstrate that backtracking is the primary mechanism by which torsional stress limits transcription and that the transcription factor GreB effectively enhances the torsional capacity of RNAP. These findings suggest a broader role for transcription factors in regulating RNAP functionality and elongation.
5. Structural visualization of RNA polymerase III transcription machineries

   https://doi.org/10.1038/s41421-018-0044-z  

   Han, Yan ; Yan, Chunli ; Fishbain, Susan ; Ivanov, Ivaylo ; He, Yuan ( July 2018 , Cell Discovery)

   RNA polymerase III (Pol III) transcription initiation requires the action of the transcription factor IIIB (TFIIIB) and is highly regulated. Here, we determine the structures of Pol III pre-initiation complexes (PICs) using single particle cryo-electron microscopy (cryo-EM). We observe stable Pol III–TFIIIB complexes using nucleic acid scaffolds mimicking various functional states, in which TFIIIB tightly encircles the upstream promoter DNA. There is an intricate interaction between TFIIIB and Pol III, which stabilizes the winged-helix domains of the C34 subunit of Pol III over the active site cleft. The architecture of Pol III PIC more resembles that of the Pol II PIC than the Pol I PIC. In addition, we also obtain a 3D reconstruction of Pol III in complex with TFIIIB using the elongation complex (EC) scaffold, shedding light on the mechanism of facilitated recycling of Pol III prior to transcription re-initiation.