Two RNA Polymerase I Subunits Control the Binding and Release of Rrn3 during Transcription

Abstract

ABSTRACT Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34 Δ weakened the binding of Rpa49 to RNA polymerase. rpa34 Δ mutants were caffeine sensitive, and the rpa34 Δ mutation was lethal in a top1 Δ mutant and in rpa14 Δ, rpa135 ( L656P ), and rpa135 ( D395N ) RNA polymerase mutants. These defects were shared by rpa49 Δ mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34 Δ mutants, but strains carrying rpa49 Δ and rpa49 - 338 :: HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30°C, failed to grow at 25°C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49 Δ and rpa49 - 338 :: HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43 - 35 , 326 , and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.