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RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways.

Original publication

DOI

10.1016/j.str.2010.11.009

Type

Journal article

Journal

Structure

Publication Date

12/01/2011

Volume

19

Pages

90 - 100

Keywords

Apoenzymes, Polydeoxyribonucleotides, Protein Multimerization, Protein Stability, Protein Structure, Quaternary, Protein Structure, Tertiary, RNA Polymerase I, RNA Polymerase III, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spectrometry, Mass, Electrospray Ionization