The A14–A43 heterodimer subunit in yeast RNA pol I and their relationship to Rpb4–Rpb7 pol II subunits

Abstract

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I-Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43-A14 pair is likely the pol I counterpart of the Rpb7-Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7-Rpb4 subunits in the three-dimensional structure of yeast pol II.T he yeast Saccharomyces cerevisiae possesses three forms of nuclear RNA polymerase (pol I, II, and III), which are distinct by their subcellular localization, chromatographic behavior, subunit composition, sensitivity to ␣-amanitine, and promoter͞template specificity.The unique essential function of pol I is to transcribe multiple ribosomal DNA units to generate the 35S ribosomal precursor (1), which is subsequently matured into the functional 18S, 5.8S, and 25S RNA species (2). Yeast pol I contains 14 subunits that include a core of five subunits (A190, A135, AC40, AC19, and ABC23) related to the ␤Ј␤␣ 2 eubacterial core enzyme (3, 4). In addition to ABC23, four subunits (ABC27, ABC14.5, ABC10␣, and ABC10␤) are shared by the three forms of enzyme (5). Finally, five other polypeptides (A49, A43, A34.5, A14, and A12.2) are subunits of pol I (6-10).Although requirement of such a complex structure is still an open question, substantial amounts of data have highlighted functional properties of subunits. The active site is carried out by the two large subunits in bacteria, archaebacteria and eukaryotes (11)(12)(13)(14). Additional studies have provided insights into the function of smaller subunits of pol I. The nonessential A34.5 subunit may help the enzyme to overcome the topological constraints imposed on rDNA by transcription (8). The dispensable A14 subunit might cooperate with A34.5 subunit in this process (9) and is important for the stabilization of subunits A43 and ABC23 within the pol I (9). A49 subunit displays ribonuclease H activity (15), whose involvement in pol I transcription is still elusive. A12.2 subunit is the pol I counterpart of pol II and pol III subunits involved in RNA cleavage activity of both enzymes (16,17), suggesting that A12.2 may be implicated in the retraction and͞or termination of pol I. Finally, the essential A43 subunit (7), ...

The RPB7 Orthologue E′ Is Required for Transcriptional Activity of a Reconstituted Archaeal Core Enzyme at Low Temperatures and Stimulates Open Complex Formation

“…The Rpb7 Ortholog EЈ Stimulates Transcription at Low Temperatures by Catalyzing Open Complex Formation-The EЈ-F complex and its eukaryotic orthologs Rpb7/Rpb4 (RNAPII), Rpc25/Rpc17 (RNAPIII), and Rpa43/Rpa14 (RNAPI) form heterodimers that are a landmark of all non-bacterial RNA polymerases (33)(34)(35)(36)(37). The structure of the Rpb7/Rpb4 complex is very similar to the archaeal EЈF dimer structure (22,17) and to the RNAPIII counterpart of these subunits, C17/C25 (37,38).…”

The Fission Yeast Protein Ker1p Is an Ortholog of RNA Polymerase I Subunit A14 in Saccharomyces cerevisiae and Is Required for Stable Association of Rrn3p and RPA21 in RNA Polymerase I

“…We found that Ker1p shows 21% identity and 27% similarity to the 126-amino acid sequence of S. cerevisiae A14 and that the N-terminal 60 amino acids of Ker1p show especially high identity (37%) and similarity (43%) to S. cerevisiae A14. The N-terminal region also shows significant identity between the A14 subunits of S. cerevisiae and C. albicans (11), and Ker1p shows 26% identity and 36% similarity to the 132-amino acid sequence of C. albicans A14. The local identity of Ker1p to the S. cerevisiae and C. albicans A14 subunits is especially high (38 and 42%, respectively) between amino acids 42 and 65 of Ker1p, and this region contains a motif that may be conserved between the S. cerevisiae and C. albicans A14 subunits (SQLKRIQR), as already suggested by Peyroche et al (11).…”

“…Because RPA21 is an ortholog of A43 of S. cerevisiae, we re-examined the homology of Ker1p to S. cerevisiae A14, which heterodimerizes with A43. Previously, A14 was suggested to have homology to a putative open reading frame of IPF1568 from Candida albicans (11), suggesting that the A14 gene family is conserved in C. albicans, although no genetic or biochemical evidence was presented. This suggestion prompted us to directly investigate the homologies among Ker1p, A14, and IPF1568 (hereafter referred to as C. albicans A14), and Fig.…”

“…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.…”

“…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 the three-dimensional structure of pol I has been deduced to be similar to that of Rpb4/Rpb7, forming an upstream interface with the C-terminal domain of Rpb1 to interact with transcription factor IIB for pol II recruitment to the pol II promoter (4) and, furthermore, playing a role in the processing of the nascent RNA transcript (17).…”