Divergent Contributions of Conserved Active Site Residues to Transcription by Eukaryotic RNA Polymerases I and II

Multisubunit RNA polymerases (msRNAPs) exhibit high sequence and structural homology, especially within their active sites, which is generally thought to result in msRNAP functional conservation. However, we show that mutations in the trigger loop (TL) in the largest subunit of RNA polymerase I (Pol I) yield phenotypes unexpected from studies of Pol II. For example, a well-characterized gain-of-function mutation in Pol II results in loss of function in Pol I (Pol II: rpb1- E1103G; Pol I: rpa190-E1224G). Studies of chimeric Pol II enzymes hosting Pol I or Pol III TLs suggest that consequences of mutations that alter TL dynamics are dictated by the greater enzymatic context and not solely the TL sequence. Although the rpa190-E1224G mutation diminishes polymerase activity, when combined with mutations that perturb Pol I catalysis, it enhances polymerase function, similar to the analogous Pol II mutation. These results suggest that Pol I and Pol II have different rate-limiting steps. [Display omitted] •RNA polymerase I poorly tolerates mutations in its trigger loop•Identical mutations result in divergent phenotypes in RNA polymerases I and II•Consequences of mutations in polymerase trigger loops depend on enzyme context•RNA polymerases I and II may have different rate-limiting steps for elongation Kaplan, Schneider, and colleagues now find that closely related eukaryotic RNA polymerases have unique enzymatic properties. Comparison of a series of mutations in the trigger loop regions of RNA polymerases I and II revealed that cells are less tolerant of mutations in RNA polymerase I. Furthermore, the properties of mutations in Pol I were different from those observed for identical mutations in Pol II. Altogether, their data suggest that high sequence conservation between related RNA polymerases does not necessarily imply functional identity.