Abstract
RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms ‘activation by cleft contraction’ and ‘hibernation by dimerization’ are unique to the regulation of this enzyme, but structure-function analysis is limited to baker’s yeast. To understand whether regulation by such strategies is specific to this model organism or conserved among species, we solve three cryo-EM structures of Pol I from Schizosaccharomyces pombe in different functional states. Comparative analysis of structural models derived from high-resolution reconstructions shows that activation is accomplished by a conserved contraction of the active center cleft. In contrast to current beliefs, we find that dimerization of the S. pombe polymerase is also possible. This dimerization is achieved independent of the ‘connector’ domain but relies on two previously undescribed interfaces. Our analyses highlight the divergent nature of Pol I transcription systems from their counterparts and suggest conservation of regulatory mechanisms among organisms.
Highlights
RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes
Two cryo-EM datasets were collected on a Titan Krios Electron Microscope equipped with Falcon III direct electron detector: one from non-crosslinked, locked nucleic acid (LNA)-containing elongation complex (EC) particles and one from BS3-crosslinked Pol I, both following size exclusion chromatography (Supplementary Table 1)
We described three single particle cryo-EM reconstructions of Schizosaccharomyces pombe (Sp) Pol I, representing the only structures of this enzyme from an organism other than Saccharomyces cerevisiae (Sc) to date
Summary
RNA polymerase (Pol) I transcribes the ribosomal RNA precursor in all eukaryotes. The mechanisms ‘activation by cleft contraction’ and ‘hibernation by dimerization’ are unique to the regulation of this enzyme, but structure-function analysis is limited to baker’s yeast. Structures solved from Sc Pol I crystals revealed inactive polymerase dimers with widely expanded active center clefts in three similar conformations[5,6,7], matching biochemical observations in extracts[8], initial and recent Electron Microscopy studies[9,10]. Such dimerization relies on a ‘connector’ domain at the C-terminus of Pol I subunit A43. Our results allow discussing the evolutionary conservation of structural features, hibernation and activation mechanisms
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