title: Mass spectrometry reveals stable modules in holo and apo RNA polymerases I and III.
abstract: 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.
authors.affiliation: Department of Chemistry, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Lane
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández-Tornero
authors.forename: Min
authors.initials: M
authors.lastname: Zhou
authors.forename: Nina
authors.initials: N
authors.lastname: Morgner
authors.forename: Denis
authors.initials: D
authors.lastname: Ptchelkine
authors.forename: Ulrich
authors.initials: U
authors.lastname: Steuerwald
authors.forename: Argyris
authors.initials: A
authors.lastname: Politis
authors.forename: Doris
authors.initials: D
authors.lastname: Lindner
authors.forename: Jelena
authors.initials: J
authors.lastname: Gvozdenovic
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gavin
authors.forename: Christoph W
authors.initials: CW
authors.lastname: Müller
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D001051:Apoenzymes / Q000737:chemistry; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012318:RNA Polymerase I / Q000737:chemistry*; D012320:RNA Polymerase III / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D001051:Apoenzymes; D011089:Polydeoxyribonucleotides; D029701:Saccharomyces cerevisiae Proteins; D012318:RNA Polymerase I; D012320:RNA Polymerase III