Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process

Complete Structural Model of Escherichia coli RNA Polymerase from a Hybrid Approach

A combination of structural approaches yields a complete atomic model of the highly biochemically characterized Escherichia coli RNA polymerase, enabling fuller exploitation of E. coli as a model for understanding transcription

Impact of template backbone heterogeneity on RNA polymerase II transcription

Variations in the sugar component (ribose or deoxyri-bose) and the nature of the phosphodiester linkage (3′-5 ′ or 2′-5 ′ orientation) have been a challenge for genetic information transfer from the very beginning of evolution. RNA polymerase II (pol II) governs the transcription of DNA into precursor mRNA in all eu-karyotic cells. How pol II recognizes DNA template backbone (phosphodiester linkage and sugar) and whether it tolerates the backbone heterogeneity re-main elusive. Such knowledge is not only important for elucidating the chemical basis of transcriptional fidelity but also provides new insights into molecular evolution. In this study, we systematically and quan-titatively investigated pol II transcriptional behaviors through different template backbone variants. We re-vealed that pol II can well tolerate and bypass sugar heterogeneity sites at the template but stalls at phos-phodiester linkage heterogeneity sites. The distinct impacts of these two backbone components on pol II transcription reveal the molecular basis of template recognition during pol II transcription and provide the evolutionary insight from the RNA world to the contemporary ‘imperfect ’ DNA world. In addition, our results also reveal the transcriptional consequences from ribose-containing genomic DNA