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

Abstract

Transcription 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.

Nucleotide Loading Modes of Human RNA Polymerase II as Deciphered by Molecular Simulations

“…The main amino acids coordinating the NTP loading processes presented in this article are conserved among eukaryotes and archaea ( Tables S6 and S7 ), advocating for a universal mechanism among the latter domains of life. Future investigation could focus on the derivation of the on- and off-pathway NTP diffusion rates along the binding macro-regions and their correlation to experimental elongation rates; downstream bubble remodeling modulation in bacterial and archaeal RNAPs [ 82 , 83 , 84 ], as well as in RNAPI and III [ 85 , 86 , 87 ]; regulation of TFIIF association [ 88 , 89 , 90 , 91 ]; and the utilization of the various NTP loading modes for different stages of the transcription cycle (initiation, pause, arrest, termination).…”

Mechanism of Transcription Termination by RNA Polymerase III Utilizes a Non-template Strand Sequence-Specific Signal Element

“…Elongation factor Spt5, which is a NusG homolog in eukaryotes, also binds in close proximity to the NT strand in the RNAP II transcription bubble (Martinez-Rucobo et al, 2011). Of further note is that termination mutagenesis screens of the second largest subunits of yeast RNAPs III and II (Kubicek et al, 2013; Shaaban et al, 1995) uncovered regions conserved in both, suggesting that their mechanisms of transcript release may be similar.…”

A methods review on use of nonsense suppression to study 3′ end formation and other aspects of tRNA biogenesis

“…A mutagenesis gain-of-function screening approach was taken for S. cerevisiae pol II; its termination signal, the poly(A) addition site, was placed in an intron of a LacZ reporter and used to screen mutagenized Rpb2 (163). Remarkably, some of the mutations uncovered are analogous to the Rpc2 lobe of pol III, in positions that interact with TFIIF subunits which share sequence homology with the RpC2 lobe-interacting domains of the pol III termination subunits, C53/37 (129, 163).…”

“…Remarkably, some of the mutations uncovered are analogous to the Rpc2 lobe of pol III, in positions that interact with TFIIF subunits which share sequence homology with the RpC2 lobe-interacting domains of the pol III termination subunits, C53/37 (129, 163). This suggests that termination by pols II and III exhibit similarities despite divergence in the cis-signals and mechanisms that initiate the process.…”