Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil

Abstract

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the ''lid'' and ''rudder,'' two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

“…1B), and biochemical data suggest that E1103G likely affects catalysis via TL dynamics, by biasing the TL toward a more closed, catalytically active conformation (6, 7). Previous ensemble measurements have shown that E1103G leads to an increase in the overall elongation rate (6,7,12), which we confirmed in our single-molecule experiments at saturating NTP in the presence of 10 pN assisting force (Fig. 1C).…”

“…The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7,(9)(10)(11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6,7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13). To explore further the interplay between elongation rate and transcriptional fidelity, we determined the effect of E1103G and H1085A substitutions on the nucleotideaddition cycle (NAC) using single-molecule assays.…”

Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation

“…6A). This is consistent with the observed twoto 4.5-fold decrease in Pol II speed measured in vitro (Malagon et al 2006). We observed a Pol II drop-off rate similar to that described previously (Jimeno-González et al 2010), but quantitative modeling excludes drop-off of Pol II during elongation as the cause for the decreased synthesis rates (Supplemental Fig.…”

“…We used a yeast strain that carries the nondisruptive point mutation N488D in the largest Pol II subunit Rpo21 (also known as Rpb1) (rpb1-N488D). This mutation slows down Pol II speed in RNA elongation assays in vitro (Malagon et al 2006) and is located near the active site (Cramer et al 2001). We subjected this strain and an isogenic wild-type strain to cDTA, and collected two biological replicates that showed a Spearman correlation of 0.99 for total and labeled Figure 3.…”

“…Strains RPB1 and rpb1-N488D (GRY 3020 and GRY 3027, respectively) were generously provided by Mikhail Kashlev (Malagon et al 2006). Genotypes of GRY 3020 and GRY 3027 are MATa, his3D1, leu2D0, lys2D0, met15D0, trp1DThisG, URA3TCMV-tTA RPO21, and MATa, his3D1, leu2D0, lys2D0, met15D0, trp1DThisG, URA3TCMV-tTA rpb1-N488D.…”