Controlled interplay between trigger loop and Gre factor in the RNA polymerase active centre

The highly processive transcription by multi-subunit RNA polymerases (RNAP) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. Backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. Here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of RNAP active centre, the trigger loop (TL), is substituted by transcription factor Gre. This substitution turns off the intrinsic TL-dependent hydrolytic activity of RNAP active centre, and exchanges it to a far more efficient Gre-dependent mechanism of RNA hydrolysis. Replacement of the TL by Gre factor occurs only in backtracked/misincorporated complexes, and not in correctly elongating complexes. This controlled switching of RNAP activities allows the processivity of elongation to be unaffected by the hydrolytic activity of Gre, while ensuring efficient proofreading of transcription and resolution of backtracked complexes

The architecture of RNA polymerase fidelity

The basis for transcriptional fidelity by RNA polymerase is not understood, but the 'trigger loop', a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A study just published in BMC Biology sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates. See research article http://www.biomedcentral.com/1741-7007/8/5

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