Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukary-otic cell growth. Here we report the 12 A ˚ cryo-electron microscopic structure for the complete 14-subunit yeast Pol I, a homology model for the core enzyme, and the crystal structure of the subcomplex A14/43. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3 0-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3 0-end trimming.

The eukaryotic RNA polymerases Pol I, Pol II, and Pol III are the central multiprotein machines that synthesize ribosomal, messenger, and transfer RNA, respectively. Here we provide a catalog of available structural information for these three enzymes. Most structural data have been accumulated for Pol II and its functional complexes. These studies have provided insights into many aspects of the transcription mechanism, including initiation at promoter DNA, elongation of the mRNA chain, tunability of the polymerase active site, which supports RNA synthesis and cleavage, and the response of Pol II to DNA lesions. Detailed structural studies of Pol I and Pol III were reported recently and showed that the active center region and core enzymes are similar to Pol II and that strong structural differences on the surfaces account for gene class-specific functions.

The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.