High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

Qiu, Chenxi; Erinne, Olivia C.; Dave, Jui M.; Cui, Ping; Jin, Hailing; Muthukrishnan, Nandhini; Tang, Leung K.; Babu, Sabareesh Ganesh; Lam, Kenny C.; Vandeventer, Paul J.; Strohner, Ralf; Brülle, Jan; Sze, Sing‐Hoi; Kaplan, Craig D.

doi:10.1101/068726

Cited by 6 publications

(10 citation statements)

References 76 publications

(191 reference statements)

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“…rpb2-Y769F exhibited strong growth defects towards MPA and Mn 2+ while we observed no effect for rpb2-P1018S ( Fig EV2A). rpb2-Y769F thus shows a growth phenotype consistent with fast RNAPII transcription mutants [20]. Interestingly, the rpb2-Y769F/P1018S double mutant exhibited mild sensitivity towards MPA compared to either single mutant ( Fig EV2A), consistent with a complementary effect on transcription speed as seen across many RNAPII active site mutations in budding yeast [27].…”

Section: Nrpb2 Y732f Accelerates Rnapii Transcription In Vivosupporting

confidence: 54%

“…To investigate the effect of NRPB2 Y732F on RNAPII transcription speed, we first tested whether the equivalent rpb2-Y769F mutant in budding yeast classifies as a fast or slow RNAPII transcription mutant by assaying its sensitivity towards mycophenolic acid (MPA) and Mn 2+ [35,36]. Budding yeast RNAPII mutants conferring enhanced catalytic activity (RNAPII fast mutants) are more sensitive towards Mn 2+ than the RNAPII slow mutants [20]. In budding yeast, RNAPII fast mutants are sensitive to MPA due to deficient expression of IMD2 gene, which counteracts the inhibition of GTP synthesis by MPA.…”

Section: Nrpb2 Y732f Accelerates Rnapii Transcription In Vivomentioning

confidence: 99%

“…For MPA and Mn 2+ growth assay, MPA (final concentration 20 mg/ml) and MnCl 2 (15 mM) were supplemented to minimal SC-Leucine medium. The yeast RNAPII mutant strains were generated by site-directed mutagenesis as previously described [20]. TSS selection of ADH1 gene was assayed by primer extension analysis.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…TSS selection of ADH1 gene was assayed by primer extension analysis. In brief, corresponding yeast strains were grown in YPD media to mid-log phase; 30 lg of isolated yeast total RNA from each indicated strains was used in primer extension analysis exactly as previously described [20,27].…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…A "gating tyrosine" in the RNAPII second largest subunit RPB2 (i.e., Y769 in budding yeast Rpb2) stacks with the first backtracked nucleotide and is proposed to prevent further backtracking [19] and is also positioned to interact with the TL when in its closed, catalysis-promoting state. Point mutations in budding yeast Rpb1 TL residues and Rpb2 TL-interacting residues alter the RNAPII elongation speed in vivo [20][21][22][23][24]. Such "kinetic RNAPII mutants" have informed greatly on the effects of altered transcription speed on gene expression and transcription-related phenotypes.…”

Section: Introductionmentioning

confidence: 99%

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Organismal benefits of transcription speed control at gene boundaries

et al. 2020

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RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto‐immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read‐through transcription. The accelerated transcription mutant NRPB2‐Y732F exhibits increased association with 5′ splice site (5′SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.

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Section: Nrpb2 Y732f Accelerates Rnapii Transcription In Vivosupporting

confidence: 54%

Section: Nrpb2 Y732f Accelerates Rnapii Transcription In Vivomentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Organismal benefits of transcription speed control at gene boundaries

et al. 2020

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Wide-ranging and unexpected consequences of altered Pol II catalytic activity in vivo

Malik

Qiu

Snavely

et al. 2016

Preprint

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14Genetic and biochemical studies have identified RNA polymerase II (Pol II) activity mutants that alter 15 catalysis, which we use as tools to investigate transcription mechanisms. Gene expression 16 consequences due to altered Pol II activity in vivo are complex. We find that alteration of Pol II catalysis 17 decreases Pol II occupancy and reporter gene expression in vivo. Both reduced and increased activity 18Pol II mutants lead to apparent reduction in elongation rate in vivo in a commonly used chromatin IP 19 assay, and we identify a confounding variable affecting interpretation of this assay. Pol II mutant effects 20 on gene expression are exacerbated with increased promoter strength and gene length. mRNA 21 degradation rates for a reporter gene are altered in Pol II mutant strains, with magnitude of half-life 22 increases correlating both with mutants' growth and gene expression defects. Prior work has suggested 23 that altered Pol II elongation sensitizes cells to nucleotide depletion. We reexamine this hypothesis and 24find that Pol II activity mutants and several elongation factor mutants respond to GTP starvation similarly 25to wild type and that putative elongation defects are not likely to drive the cellular response to limiting 26 GTP. 27 28

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Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Amundsen

Taylor

Smith

2020

Preprint

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Life-saving repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5' GCTGGTGG 3') recognized during DNA unwinding. Our previous studies led to a signal transduction model in which the Chi-bound RecC subunit signals RecD, a fast helicase, to stop; RecD then signals RecB, a slower helicase, to swing its nuclease domain into position to cut DNA at Chi and load RecA protein onto the newly generated 3'-ended single-stranded DNA. Here, we report a set of mutants blocked at each step in this pathway. The mutants have helicase and degradative nuclease activities, but most have lost or reduced coordination by Chi and have reduced recombination and DNA repair proficiency. The mutant alterations are widely scattered throughout the three subunits of RecBCD; each alters the intimate contact between two subunits in the crystal or cryoEM structures, supporting the signal transduction model. Similar large conformational changes in other vital enzymes, such as DNA and RNA polymerases, may make RecBCD a paradigm for elucidating their control by the nucleic acid sites they recognize. Author summaryRepair of broken DNA is essential for life. Faithful repair occurs by homologous genetic recombination, which also generates new gene allele combinations important for evolution. In bacteria the RecBCD helicase-nuclease enzyme is essential for most DNA break repair and is regulated by special DNA sites (Chi recombination hotspots). As it unwinds DNA, RecBCD's activities are dramatically altered at Chi to prepare the broken DNA for interaction with intact DNA to finish repair. Here, we describe RecBCD mutants that respond weakly, or not at all, to Chi; the alterations are scattered widely throughout the enzyme, supporting our previous model of large conformational changes of RecBCD at Chi. Similar methods may help understand how other large enzyme complexes are regulated.

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High-resolution phenotypic landscape of the RNA Polymerase II trigger loop

Abstract: 22 23The active site of multicellular RNA polymerases have a "trigger loop" (TL) that

Cited by 6 publications

References 76 publications

Organismal benefits of transcription speed control at gene boundaries

Organismal benefits of transcription speed control at gene boundaries

Wide-ranging and unexpected consequences of altered Pol II catalytic activity in vivo

Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

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