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.     

Rpb4, a Subunit of RNA Polymerase II, Enables the Enzyme To Transcribe at Temperature Extremes In Vitro

Rpb4 is a subunit of Saccharomyces cerevisiae RNA polymerase II (Pol II). It associates with the polymerase preferentially in stationary phase and is essential for some stress responses. Using the promoter-independent initiation and chain elongation assay, we monitored Pol II enzymatic activity in cell extracts. We show here that Rpb4 is required for the polymerase activity at temperature extremes (10 and 35°C). In contrast, at moderate temperature (23°C) Pol II activity is independent of Rpb4. These results are consistent with the role previously attributed to Rpb4 as a subunit whose association with Pol II helps Pol II to transcribe during extreme temperatures. The enzymatic inactivation of Pol II lacking Rpb4 at the nonoptimal temperature was prevented by the addition of recombinant Rpb4 produced in Escherichia coli prior to the in vitro reaction assay. This finding suggests that modification of Rpb4 is not required for its functional association with the other Pol II subunits. Sucrose gradient and immunoprecipitation experiments demonstrated that Rpb4 is present in the cell in excess over the Pol II complex during all growth phases. Nevertheless, the rescue of Pol II activity at the nonoptimal temperature by Rpb4 is possible only when cell extracts are obtained from postlogarithmic cells, not from logarithmically growing cells. This result suggests that Pol II molecules should be modified in order to recruit Rpb4; the portion of the modified Pol II molecules is small during logarithmic phase and becomes predominant in stationary phase.