Crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe RNA polymerase III

Eukaryotic RNA polymerase III (Pol III) is a multisubunit enzyme responsible for transcribing tRNA, 5S rRNA, and several small RNAs. Of the 17 subunits in Pol III, the C17 (Rpc17) and C25 (Rpc25) subunits form a stable subcomplex that protrudes from the core polymerase. In this study, we determined the crystal structure of the C17/25 subcomplex from Schizosaccharomyces pombe. The subcomplex adopts an elongated shape, and each subunit has two domains. The two subunits in the subcomplex are tightly packed and extensively interact, with a contact area of 2080 Ų. The overall conformation of S. pombe C17/25 is considerably different from the previously reported structure of C17/25 from Saccharomyces cerevisiae, with respect to the position of the C17 HRDC domain, a helix bundle essential for cell viability. In contrast, the S. pombe C17/25 structure is quite similar to those of the Pol II and archaeal counterparts, Rpb4/7 and RpoE/F, respectively, despite the low sequence similarity. A phylogenetic comparison of the C17 subunits among eukaryotes revealed that they can be classified into three groups, according to the length of the interdomain linker. S. pombe C17, as well as Rpb4 and RpoF, belongs to the largest group, with the short linker. On the other hand, S. cerevisiae C17 belongs to the smallest group, with the long linker, which probably enables the subcomplex to assume the alternative conformation.     

Structural biology of RNA polymerase III: mass spectrometry elucidates subcomplex architecture

RNA polymerases (Pol) II and III synthesize eukaryotic mRNAs and tRNAs, respectively. The crystal structure of the 12 subunit Pol II is known, but only limited structural information is available for the 17 subunit Pol III. Using mass spectrometry (MS), we correlated masses of Pol II complexes with the Pol II structure. Analysis of Pol III showed that the complete enzyme contains a single copy of each subunit and revealed a 15 subunit form lacking the Pol III-specific subcomplex C53/37. DMSO treatment dissociated the C17/25 heterodimer of Pol III, confirming a peripheral location as its counterpart in Pol II. Tandem MS revealed the Pol III-specific subunits C82 and C34 dissociating as a heterodimer. C11 was retained, arguing against a stable trimeric subcomplex, C53/37/11. These data suggest that Pol III consists of a 10 subunit Pol II-like core; the peripheral heterodimers C17/25, C53/37, and C82/34; and subunit C31, which bridges between C82/34, C17/25, and the core.     

Crosslinking-MS analysis reveals RNA polymerase I domain architecture and basis of rRNA cleavage

RNA polymerase (Pol) I contains a 10-subunit catalytic core that is related to the core of Pol II and includes subunit A12.2. In addition, Pol I contains the heterodimeric subcomplexes A14/43 and A49/34.5, which are related to the Pol II subcomplex Rpb4/7 and the Pol II initiation factor TFIIF, respectively. Here we used lysine-lysine crosslinking, mass spectrometry (MS) and modeling based on five crystal structures, to extend the previous homology model of the Pol I core, to confirm the location of A14/43 and to position A12.2 and A49/34.5 on the core. In the resulting model of Pol I, the C-terminal ribbon (C-ribbon) domain of A12.2 reaches the active site via the polymerase pore, like the C-ribbon of the Pol II cleavage factor TFIIS, explaining why the intrinsic RNA cleavage activity of Pol I is strong, in contrast to the weak cleavage activity of Pol II. The A49/34.5 dimerization module resides on the polymerase lobe, like TFIIF, whereas the A49 tWH domain resides above the cleft, resembling parts of TFIIE. This indicates that Pol I and also Pol III are distantly related to a Pol II-TFIIS-TFIIF-TFIIE complex.     

Functional architecture of RNA polymerase I

Synthesis of ribosomal RNA (rRNA) by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic 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'-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2 and, apparently, enables ribosomal RNA proofreading and 3'-end trimming.