Eukaryotes express at least three nuclear DNA-dependent RNA polymerases (Pols) responsible for synthesizing all RNA required by the cell. Despite sharing structural homology, they have functionally diverged to suit their distinct cellular roles. Although the Pols have been studied extensively, direct comparison of their enzymatic properties is difficult since studies are often conducted under disparate experimental conditions and techniques. Here, we directly compare and reveal functional differences between Saccharomyces cerevisiae Pols I and II using a series of quantitative in vitro transcription assays. We find that Pol I single and multi-nucleotide addition rate constants are faster than those of Pol II. Pol I elongation complexes (ECs) are less stable than Pol II ECs, and Pol I is more error prone than Pol II. Collectively, these data show that the enzymatic properties of the Pols have diverged over the course of evolution, optimizing these enzymes for their unique cellular responsibilities.
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.
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