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1. A remodeled RNA polymerase II complex catalyzing viroid RNA-templated transcription

   https://doi.org/10.1371/journal.ppat.1010850  

   Dissanayaka Mudiyanselage, Shachinthaka D.; Ma, Junfei; Pechan, Tibor; Pechanova, Olga; Liu, Bin; Wang, Ying (September 2022, PLOS Pathogens)

   Daròs, José-Antonio (Ed.)

   Viroids, a fascinating group of plant pathogens, are subviral agents composed of single-stranded circular noncoding RNAs. It is well-known that nuclear-replicating viroids exploit host DNA-dependent RNA polymerase II (Pol II) activity for transcription from circular RNA genome to minus-strand intermediates, a classic example illustrating the intrinsic RNA-dependent RNA polymerase activity of Pol II. The mechanism for Pol II to accept single-stranded RNAs as templates remains poorly understood. Here, we reconstituted a robust in vitro transcription system and demonstrated that Pol II also accepts minus-strand viroid RNA template to generate plus-strand RNAs. Further, we purified the Pol II complex on RNA templates for nano-liquid chromatography-tandem mass spectrometry analysis and identified a remodeled Pol II missing Rpb4, Rpb5, Rpb6, Rpb7, and Rpb9, contrasting to the canonical 12-subunit Pol II or the 10-subunit Pol II core on DNA templates. Interestingly, the absence of Rpb9, which is responsible for Pol II fidelity, explains the higher mutation rate of viroids in comparison to cellular transcripts. This remodeled Pol II is active for transcription with the aid of TFIIIA-7ZF and appears not to require other canonical general transcription factors (such as TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and TFIIS), suggesting a distinct mechanism/machinery for viroid RNA-templated transcription. Transcription elongation factors, such as FACT complex, PAF1 complex, and SPT6, were also absent in the reconstituted transcription complex. Further analyses of the critical zinc finger domains in TFIIIA-7ZF revealed the first three zinc finger domains pivotal for RNA template binding. Collectively, our data illustrated a distinct organization of Pol II complex on viroid RNA templates, providing new insights into viroid replication, the evolution of transcription machinery, as well as the mechanism of RNA-templated transcription. 

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2. Crystal structure of an RNA/DNA strand exchange junction

   https://doi.org/10.1371/journal.pone.0263547  

   Cofsky, Joshua C.; Knott, Gavin J.; Gee, Christine L.; Doudna, Jennifer A. (April 2022, PLOS ONE)

   Kursula, Petri (Ed.)

   Short segments of RNA displace one strand of a DNA duplex during diverse processes including transcription and CRISPR-mediated immunity and genome editing. These strand exchange events involve the intersection of two geometrically distinct helix types—an RNA:DNA hybrid (A-form) and a DNA:DNA homoduplex (B-form). Although previous evidence suggests that these two helices can stack on each other, it is unknown what local geometric adjustments could enable A-on-B stacking. Here we report the X-ray crystal structure of an RNA-5′/DNA-3′ strand exchange junction at an anisotropic resolution of 1.6 to 2.2 Å. The structure reveals that the A-to-B helical transition involves a combination of helical axis misalignment, helical axis tilting and compression of the DNA strand within the RNA:DNA helix, where nucleotides exhibit a mixture of A- and B-form geometry. These structural principles explain previous observations of conformational stability in RNA/DNA exchange junctions, enabling a nucleic acid architecture that is repeatedly populated during biological strand exchange events. 

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3. Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I

   https://doi.org/10.1074/jbc.RA119.011354  

   Scull, Catherine E.; Clarke, Andrew M.; Lucius, Aaron L.; Schneider, David Alan (January 2020, Journal of Biological Chemistry)

   null (Ed.)

   The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo . Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro . 

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4. Evaluating two steps in transcription using a fluorescence‐based electrophoretic mobility shift assay

   https://doi.org/10.1002/bmb.21708  

   Singh, Anoushka; Miller, Ryan C.; Archuleta, Stephen R.; Kugel, Jennifer F. (January 2023, Biochemistry and Molecular Biology Education)

   Abstract Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence‐based electrophoretic mobility shift assay (EMSA) and commercially availableEscherichia coliRNAP. As an inquiry‐driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions. 

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5. The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex

   https://doi.org/10.1073/pnas.2207581119  

   Marshall, Craig J; Qayyum, M Zuhaib; Walker, Julie E; Murakami, Katsuhiko S; Santangelo, Thomas J (August 2022, Proceedings of the National Academy of Sciences)

   Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta–TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms. 

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