Specific Defects in Different Transcription Complexes Compensate for the Requirement of the Negative Cofactor 2 Repressor in Saccharomyces cerevisiae

Abstract

Negative cofactor 2 (NC2) has been described as an essential and evolutionarily conserved transcriptional repressor, although in vitro and in vivo experiments suggest that it can function as both a positive and a negative effector of transcription. NC2 operates by interacting with the core promoter and components of the basal transcription machinery, like the TATA-binding protein (TBP). In this work, we have isolated mutants that suppress the growth defect caused by the depletion of NC2. We have identified mutations affecting components of three different complexes involved in the control of basal transcription: the mediator, TFIIH, and RNA pol II itself. Mutations in RNA pol II include both overexpression of truncated forms of the two largest subunits (Rpb1 and Rpb2) and reduced levels of these proteins. Suppression of NC2 depletion was also observed by reducing the amounts of the mediator essential components Nut2 and Med7, as well as by deleting any of the nonessential mediator components, except Med2, Med3, and Gal11 subunits. Interestingly, the Med2/Med3/Gal11 triad forms a submodule within the mediator tail. Our results support the existence of different components within the basic transcription complexes that antagonistically interact with the NC2 repressor and suggest that the correct balance between the activities of specific positive and negative components is essential for cell growth.

Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis

“…In order to introduce genomic changes into a ⌬rtp1 mutant background, the heterozygous ⌬rtp1/RTP1 diploid was transformed and sporulated to obtain the strain with the desired genotype. The genetic screen to isolate the suppressors of NC2 has been previously described (13).…”

“…To construct pGAD-NUP116, an MfeI-NsiI fragment from pGST-Nup116 was cloned into the EcoRI-PstI sites of pGAD-C2 (21). YEp-Rpb2t has been previously described (13). The YEp-Rpb2t-TAP plasmid was made by introducing the TAP tag into the EcoRI site of RPB2 in plasmid YEp-Rpb2t.…”

“…The interaction between Rtp1p and Rpb2p prompted us to revise the effect that the expression of a truncated allele of RPB2 (named rpb2t) could have on RNA pol II localization. The expression of Rpb2tp, from a 2m-based plasmid, as occurs with the deletion of genes RTP1 and IWR1, suppresses the growth defects caused by the depletion of NC2 components (13). We hypothesized that the suppressing effect of the truncated Rpb2p subunit could be caused by an impairment of RNA pol II biogenesis through the squelching of Rtp1p.…”

“…This protein was identified in a genetic screen for suppressors of the growth defect caused by depletion of the transcription repressor NC2 (13,14) and in high-throughput MS screens of yeast as a protein that interacts with RNA pol II (15,16). Deletion of IWR1 has major effects on the cell transcription profile (14,17), although Iwr1p has not been associated with RNA pol II when this enzyme is recruited to the promoter of active genes (14).…”

Rbs1, a New Protein Implicated in RNA Polymerase III Biogenesis in Yeast Saccharomyces cerevisiae

“…Npa3, the yeast homolog of GPN1, is required for nuclear localization of yeast Pol II and binds it in a GTP-dependent manner (13), which argues that the mechanism involved in the subcellular localization of Pol II requires the catalytic function of GNP proteins and is conserved from yeast to mammals (14). Two other proteins involved in Pol II biogenesis, Iwr1 and Rtp1, were identified in genetic screens for suppressors of the growth defect caused by depletion of NC2, a negative regulator of mRNA transcription (15,16). Iwr1 binds yeast Pol II in the active-center cleft between the two largest subunits, possibly facilitating or sensing complete Pol II assembly in the cytoplasm.…”