bRecent papers have provided insight into the cytoplasmic assembly of RNA polymerase II (RNA pol II) and its transport to the nucleus. However, little is known about the mechanisms governing its nuclear assembly, stability, degradation, and recycling. We demonstrate that the foot of RNA pol II is crucial for the assembly and stability of the complex, by ensuring the correct association of Rpb1 with Rpb6 and of the dimer Rpb4-Rpb7 (Rpb4/7). Mutations at the foot affect the assembly and stability of the enzyme, a defect that is offset by RPB6 overexpression, in coordination with Rpb1 degradation by an Asr1-independent mechanism. Correct assembly is a prerequisite for the proper maintenance of several transcription steps. In fact, assembly defects alter transcriptional activity and the amount of enzyme associated with the genes, affect C-terminal domain (CTD) phosphorylation, interfere with the mRNA-capping machinery, and possibly increase the amount of stalled RNA pol II. In addition, our data show that TATA-binding protein (TBP) occupancy does not correlate with RNA pol II occupancy or transcriptional activity, suggesting a functional relationship between assembly, Mediator, and preinitiation complex (PIC) stability. Finally, our data help clarify the mechanisms governing the assembly and stability of RNA pol II. RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs but contributes less than 10% of the total RNA present in growing cells (1). It consists of 12 protein subunits with a heterodimeric subcomplex of subunits Rpb4 and Rpb7 (Rpb4/7). The catalytic core of the bacterial and eukaryotic enzymes is highly conserved through evolution. However, only five subunits have bacterial homologs (Rpb1, Rpb2, Rpb3, Rpb6, and Rpb11); the others are common to archaea but have no eubacterial homologs (2, 3). The RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides, including general transcription factors, coregulators, and specific transcription activators as well as repressors (1).Many studies have contributed to the knowledge of physical interactions between RNA pol II and transcriptional regulators and have enabled the identification of regions that are important for transcription, from initiation to mRNA export (2, 4-12). In addition, we have recently reported the existence of five "conserved domains," located at the surface of the structure of the complex, with poor or no conservation in their paralogs in RNA polymerases I (Rpa190 and Rpa135) and III (Rpc160 and Rpc128) and in their homologs in archaea and bacteria and demonstrate that all of them make contact with transcriptional regulators (10).One of these regions corresponds to the foot domain (2, 10), which, in cooperation with the "lower jaw," the "assembly" domain, and the "cleft" regions, constitutes the "shelf" module of RNA pol II, which might contribute to the rotation of the DNA as it advances toward the active center (2,8). This domain, conserv...
Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-β25/26 (prolonging the Bridge helix) and Rpb1-α44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Δ or its RNA polymerase I rpa12Δ counterpart. Rpb1-β25/26, Rpb1-α44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-α46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5–Rpb1 binding and the Rpb9–Rpb1 interaction is to hold the Bridge helix, the Rpb1-α44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.
RNA polymerase (pol) II establishes many protein-protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the "foot" domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxyterminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/ or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme.I N eukaryotes as in archaea, bacteria, chloroplasts, some mitochondria, and nucleocytoplasmic DNA viruses, transcription is ensured by heteromultimeric DNA-dependent RNA polymerases (Thuriaux and Sentenac 1992;Vassylyev et al. 2002;Werner and Weinzierl 2002;Iyer et al. 2006). RNA polymerase II (RNA pol II) produces all mRNAs and many noncoding RNAs. Although it transcribes most of the nuclear genome, it contributes ,10% of the total RNA present in growing cells (Hahn 2004). To transcribe a gene, RNA pol II requires the action of general transcription factors, coregulators, specific transcription activators, and repressors. In fact, the RNA pol II transcription machinery is the most complex of those associated with the three RNA polymerases, with a total of nearly 60 polypeptides (Hahn 2004).Knowledge of both the architecture making up this complex and the function of its different parts is essential to understand their role in the different transcription steps (Cramer 2006;Zaros et al. 2007;Venters and Pugh 2009). Structural data gathered over the last few years on Saccharomyces cerevisiae RNA pol II have provided a detailed map of the physical interactions between the different subunits, establishing regions that are important for transcription (Cramer et al. 2001;Bushnell et al. 2002;Armache et al. 2003;Meyer et al. 2009). Notably, recent work has contributed to the understanding of how RNA pol II amino acid regions or subunits are involved in the contact with transcriptional regulators such as TFIIS, TFIIB, TFIIE, TFIIF, or Mediator, among others, although the data are sometimes imprecise or controversial (Guglielmi et al. 2004;Chadick and Asturias 2005;Chen et al. 2007;Meyer et al. 2009;Kostrewa et al. 2009).A major question that remains unexplored is the identification of domains of RNA pol II that could be involved in the interaction with elements of the transcriptional machinery and that could participate in coordinating with them. The...
The mechanism of action of 6AU, a growth inhibitor for many microorganisms causing depletion of intracellular nucleotide pools of GTP and UTP, is not well understood. To gain insight into the mechanisms leading to 6AU resistance, and in an attempt to uncover novel genes required for this resistance, we undertook a high-copy-number suppressor screening to identify genes whose overexpression could repair the 6AU(S) growth defect caused by rpb1 mutations in Saccharomyces cerevisiae. We have identified SNG1 as a multicopy suppressor of the 6AU(S) growth defect caused by the S. cerevisiae rpb1 mutant. The mechanism by which Sng1 causes 6AU resistance is independent of the transcriptional elongation and of the nucleotide-pool regulation through Imd2 and Ura2, as well as of the Ssm1-mediated 6AU detoxification. This resistance to 6AU is not extended to other uracil analogues, such as 5-fluorouracil, 5FU. In addition, our results suggest that 6AU enters S. cerevisiae cells through the uracil permease Fur4. Our results demonstrate that Sng1 is localised in the plasma membrane and evidence SNG1 and FUR4 genes as determinants of resistance and susceptibility to this inhibitory compound, respectively. Taken together, these results show new mechanisms involved in the resistance and susceptibility to 6AU.
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