We obtained an 11 subunit model of RNA polymerase (Pol) III by combining a homology model of the nine subunit core enzyme with a new X-ray structure of the subcomplex C17/25. Compared to Pol II, Pol III shows a conserved active center for RNA synthesis but a structurally different upstream face for specific initiation complex assembly during promoter selection. The Pol III upstream face includes a HRDC domain in subunit C17 that is translated by 35 A and rotated by 150 degrees compared to its Pol II counterpart. The HRDC domain is essential in vivo, folds independently in vitro, and, unlike other HRDC domains, shows no indication of nucleic acid binding. Thus, the HRDC domain is a functional module that could account for the role of C17 in Pol III promoter-specific initiation. During elongation, C17/25 may bind Pol III transcripts emerging from the adjacent exit pore, because the subcomplex binds to tRNA in vitro.

Yeast RNA polymerase (Pol) II consists of a 10-subunit core enzyme and the Rpb4/7 subcomplex, which is dispensable for catalytic activity and dissociates in vitro. To investigate whether Rpb4/7 is an integral part of DNA-associated Pol II in vivo, we used chromatin immunoprecipitation coupled to high resolution tiling microarray analysis. We show that the genome-wide occupancy profiles for Rpb7 and the core subunit Rpb3 are essentially identical. Thus, the complete Pol II associates with DNA in vivo, consistent with functional roles of Rpb4/7 throughout the transcription cycle. Pol2 II is the enzyme responsible for mRNA synthesis during transcription of protein-coding genes in eukaryotic cells. The structure of the complete 12-subunit Pol II is known and consists of a 10-subunit core enzyme, which includes the active center, and the peripheral heterodimer of subunits Rpb4 and Rpb7 (Rpb4/7 subcomplex) (1-4). Whereas Rpb7 is essential for viability of the yeast Saccharomyces cerevisiae, Rpb4 is not, but it becomes essential at temperature extremes (5). In vitro, yeast Rpb4/7 is required for transcription initiation, can dissociate from Pol II, and is dispensable for catalytic RNA elongation (6). Rpb4/7 binds singlestranded nucleic acids and mediates a step during initiation subsequent to promoter DNA binding (7). These observations are consistent with the idea that Rpb4/7 is present during initiation at promoters but then dissociates from Pol II.However, evidence has accumulated for additional functional roles of Rpb4/7 during transcription. Rpb7 remains associated with early elongation complexes (8) and binds Nrd1, a protein involved in RNA 3Ј-end processing (9). Rpb4/7 can be cross-linked to the transcribed region, and loss of Rpb4 decreases the association with 3Ј-processing factors and alters usage of the polyadenylation site at a tested gene (10). Thus, recent data suggest that Rpb4/7 is an integral part of the Pol II enzyme and is required not only for initiation but also for 3Ј-RNA processing at the end of transcription.To investigate whether Rpb4/7 generally associates with Pol II in vivo, we carried out chromatin immunoprecipitation in yeast coupled to tiling microarray analysis at a technical resolution of 32 base pairs. We demonstrate that the occupancy profiles of Rpb7 and the Pol II core subunit Rpb3 are virtually identical, showing that the complete Pol II, including Rpb4/7, associates with DNA genome-wide. These data are consistent with functional roles of Rpb4/7 throughout the transcription cycle. EXPERIMENTAL PROCEDURESYeast Strains-Experiments were performed with S. cerevisiae strains W303 wild type (MATa,15,112, GALϩ) and W303 carrying a tandem affinity purification (TAP) tag sequence fused to the C terminus of the gene encoding Rpb3 (MATa,

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