Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34⌬ weakened the binding of Rpa49 to RNA polymerase. rpa34⌬ mutants were caffeine sensitive, and the rpa34⌬ mutation was lethal in a top1⌬ mutant and in rpa14⌬, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49⌬ mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34⌬ mutants, but strains carrying rpa49⌬ and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30°C, failed to grow at 25°C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49⌬ and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.Fast-growing Saccharomyces cerevisiae and Schizosaccharomyces pombe cells mobilize some 70% of their transcriptional capacity to produce the 6.8-kb precursor of the 18S, 5.8S, and 25S rRNAs by transcribing the highly repeated ribosomal DNA (rDNA) locus (44, 50). Genetic studies (43) have established that this is the only role or, at least, the only essential role of yeast RNA polymerase I (Pol I). rDNA transcription starts with the binding of Pol I to its specificity factor Rrn3 (7, 39-41, 48, 57). The Rrn3-Pol I dimer is then directed to a preinitiation complex residing at the rDNA promoter region. This complex combines the TATA box-binding protein (also operating in the Pol II and Pol III systems) with the core and upstream activation factors that are specific to the Pol I system (31, 32). In mammals, Pol I first binds transcription initiation factor IA, akin to Rrn3 and functionally interchangeable with that protein in vivo (7,40,41). The Pol I-transcription initiation factor IA dimer is then targeted to the rDNA promoter by interacting with SL1, a factor made of the TATA box-binding protein associated with four protein subunits, TAF I 95, TAF I 68, TAF I 48, and TAF I 41 (11,22,23,40,59); TAF I 68 is distantly related to the yeast core factor at the level of the core factor's Rrn7/TAF I 68 subunit (9). Pol I is also associated with the upstream binding factor (UBF; mammals) and Hmo1 (yeast) HMG box proteins, which stimulate rDNA t...
Saccharomyces cerevisiae A49 and mouse PAF53 are subunits specific to RNA polymerase I (Pol I) in eukaryotes. It has been known that Pol I without A49 or PAF53 maintains non-specific transcription activities but a molecular role(s) of A49 (and PAF53) remains totally unknown. We studied the fission yeast gene encoding a protein of 415 amino acids exhibiting 30% and 19% identities to A49 and PAF53, respectively. We designate the corresponding protein RPA51 and gene encoding it rpa51 + since the gene encodes a Pol I subunit and an apparent molecular mass of the protein is 51 kDa. rpa51+ is required for cell growth at lower but not at higher temperatures and is able to complement S. cerevisiae rpa49 ∆ mutation, indicating that RPA51 is a functionally-conserved subunit of Pol I between the budding yeast and the fission yeast. Deletion analysis of rpa51 + shows that only two-thirds of the C-terminal region are required for the function. Transcripts analysis in vivo and in vitro shows that RPA51 plays a general role for maximizing transcription of rDNA whereas it is dispensable for non-specific transcription. We also found that RPA51 associates significantly with Pol I in the stationary phase, suggesting that Pol I inactivation in the stationary phase of yeast does not result from the RPA51 dissociation.
A heterodimer formed by the A14 and A43 subunits of RNA polymerase (pol) I in Saccharomyces cerevisiae is proposed to correspond to the Rpb4/Rpb7 and C17/C25 heterodimers in pol II and pol III, respectively, and to play a role(s) in the recruitment of pol I to the promoter. However, the question of whether the A14/A43 heterodimer is conserved in eukaryotes other than S. cerevisiae remains unanswered, although both Rpb4/Rpb7 and C17/C25 are conserved from yeast to human. To address this question, we have isolated a Schizosaccharomyces pombe gene named ker1 ؉ using a yeast twohybrid system, including rpa21 ؉ , which encodes an ortholog of A43, as bait. Although no homolog of A14 has previously been found in the S. pombe genome, functional characterization of Ker1p and alignment of Ker1p and A14 showed that Ker1p is an ortholog of A14. Disruption of ker1 ؉ resulted in temperature-sensitive growth, and the temperature-sensitive deficit of ker1⌬ was suppressed by overexpression of either rpa21 ؉ or rrn3 ؉ , which encodes the rDNA transcription factor Rrn3p, suggesting that Ker1p is involved in stabilizing the association of RPA21 and Rrn3p in pol I. We also found that Ker1p dissociated from pol I in post-logphase cells, suggesting that Ker1p is involved in growthdependent regulation of rDNA transcription.There are three distinct types of eukaryotic nuclear RNA polymerases: RNA polymerase (pol) 1 I, pol II, and pol III. Among eukaryotic organisms, the structure and function of RNA polymerases in Saccharomyces cerevisiae have been studied fairly extensively (1-4). S. cerevisiae pol I consists of 14 subunits. The core structure contains 10 subunits (A190, A135, AC40, AC19, Rpb5, Rpb6, Rpb8, Rpb10, Rpb12, and A12.2) and is believed to be sufficient for nonspecific transcription, but not for accurate initiation of transcription (5). In fact, pol I requires four specific subunits (A49, A43, A34.5, and A14) for specific transcription of rDNA. A43 is also essential for cell growth (6), whereas A49 (7), A34.5 (8), and A14 (9) are dispensable.Much attention has recently been focused on the A14 and A43 subunits in view of the structural and functional conservation of these two subunits in eukaryotes. A43 is conserved in a variety of eukaryotes (10) and shows amino acid sequence similarity to Rpb7 (a specific subunit of pol II), C25 (a specific subunit of pol III), and RpoE (a subunit of archaeal RNA polymerases) across multiple RNA polymerases (11). Furthermore, A43 forms a heterodimer with A14 that is similar to the Rpb4/Rpb7 (11, 12), C17/C25 (13), and RpoF/RpoE (14) heterodimers in pol II, pol III, and archaeal RNA polymerases, respectively. It should be noted that Rpb4, C17, and RpoF have mutual sequence similarity and are grouped into a gene family, but no obvious homolog of A14 has been found in available data bases. A14 and Rpb4 are required for the stable assembly of A43 and Rpb7, respectively, in their respective RNA polymerases, suggesting a functional similarity of A14 to Rpb4 (5,11,15,16). The position of A14/A43 in...
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