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A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases.

ABSTRACT: Eukaryotic cells express at least three nuclear RNA polymerases (Pols), each with a unique set of gene targets. Though these enzymes are homologous, there are many differences among the Pols. In this study, a novel assay for Pol I transcription elongation was developed to probe enzymatic differences among the Pols. In Saccharomyces cerevisiae, a mutation in the universally conserved hinge region of the trigger loop, E1103G, induces a gain of function in the Pol II elongation rate, whereas the corresponding mutation in Pol I, E1224G, results in a loss of function. The E1103G Pol II mutation stabilizes the closed conformation of the trigger loop, promoting the catalytic step, the putative rate-limiting step for Pol II. In single-nucleotide and multinucleotide addition assays, we observe a decrease in the rate of nucleotide addition and dinucleotide cleavage activity by E1224G Pol I and an increase in the rate of misincorporation. Collectively, these data suggest that Pol I is at least in part rate-limited by the same step as Pol II, the catalytic step.

SUBMITTER: Scull CE

PROVIDER: S-EPMC6600827 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases.

Scull Catherine E CE Ingram Zachariah M ZM Lucius Aaron L AL Schneider David A DA

Biochemistry 20190405 16

Eukaryotic cells express at least three nuclear RNA polymerases (Pols), each with a unique set of gene targets. Though these enzymes are homologous, there are many differences among the Pols. In this study, a novel assay for Pol I transcription elongation was developed to probe enzymatic differences among the Pols. In Saccharomyces cerevisiae, a mutation in the universally conserved hinge region of the trigger loop, E1103G, induces a gain of function in the Pol II elongation rate, whereas the co ...[more]

PMID: 30912638

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Project description:BACKGROUND: The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. RESULTS: We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the ?C helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. CONCLUSIONS: We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and propose specific hypotheses for experimental investigation. The apicomplexan-specific kinase features reported in this study can be used in the design of selective kinase inhibitors.

Dataset Information

A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases.

Publications

A Novel Assay for RNA Polymerase I Transcription Elongation Sheds Light on the Evolutionary Divergence of Eukaryotic RNA Polymerases.

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