Abstract
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.
Highlights
Cellular DNA-dependent RNA polymerases likely balance fidelity in substrate selection with synthesis speed to achieve appropriate transcriptome content and regulation in vivo
We present analysis of the RNA Polymerase II (Pol II) trigger loop (TL) from the model eukaryote Saccharomyces cerevisiae
We find that in vivo growth phenotypes correlate with severity of transcriptional defects and that changing Pol II activity to either faster or slower than wild type causes specific transcription defects
Summary
Cellular DNA-dependent RNA polymerases likely balance fidelity in substrate selection with synthesis speed to achieve appropriate transcriptome content and regulation in vivo. To mobile domains of other classes of nucleic acid polymerases, the TL undergoes conformational changes in conjunction with the presence of an NTP substrate complementary to the DNA template (matched) in the msRNAP active site [1,3]. These conformational changes are proposed to link TLsubstrate interactions to preferential catalysis of correctly matched substrates over mismatched substrates. In addition to effects on phosphodiester bond catalysis, the TL has been implicated in polymerase pausing, intrinsic cleavage of RNA and translocation [7,8,9,16,17]
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