Abstract
Eukaryotes express three nuclear RNA polymerases (Pols I, II, and III) that are essential for cell survival. Despite extensive investigation of the three Pols, significant knowledge gaps regarding their biochemical properties remain because each Pol has been evaluated independently under disparate experimental conditions and methodologies. To advance our understanding of the Pols, we employed identical in vitro transcription assays for direct comparison of their elongation rates, elongation complex (EC) stabilities, and fidelities. Pol I is the fastest, most likely to misincorporate, forms the least stable EC, and is most sensitive to alterations in reaction buffers. Pol II is the slowest of the Pols, forms the most stable EC, and negligibly misincorporated an incorrect nucleotide. The enzymatic properties of Pol III were intermediate between Pols I and II in all assays examined. These results reveal unique enzymatic characteristics of the Pols that provide new insights into their evolutionary divergence.