RNA Polymerase II Mutations Conferring Defects in Poly(A) Site Cleavage and Termination inSaccharomyces cerevisiae

Abstract

AbstractTranscription termination by RNA polymerase (Pol) II is an essential but poorly understood process. In eukaryotic nuclei, the 3′ ends of mRNAs are generated by cleavage and polyadenylation, and the same sequence elements that specify that process are required for downstream release of the polymerase from the DNA. Although Pol II is known to bind proteins required for both events, few studies have focused on Pol II mutations as a means to uncover the mechanisms that couple polyadenylation and termination. We performed a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutations in the N-terminal half of Rpb2, the second largest Pol II subunit, that conferred either a decreased or increased response to a well-characterized poly(A) site. Most of the mutant alleles encoded substitutions affecting either surface residues or conserved active site amino acids at positions important for termination by other RNA polymerases. Reverse transcription polymerase chain reaction experiments revealed that transcript cleavage at the poly(A) site was impaired in both classes of increased readthrough mutants. Transcription into downstream sequences beyond where termination normally occurs was also probed. Although most of the tested readthrough mutants showed a reduction in termination concomitant with the reduced poly(A) usage, these processes were uncoupled in at least one mutant strain. Several rpb2 alleles were found to be similar or identical to published mutants associated with defective TFIIF function. Tests of these and additional mutations known to impair Rpb2−TFIIF interactions revealed similar decreased readthrough phenotypes, suggesting that TFIIF may have a role in 3′ end formation and termination.