title: Lysine methylation mapping of crenarchaeal DNA-directed RNA polymerases by collision-induced and electron-transfer dissociation mass spectrometry.
abstract: Enzymatic machineries fundamental for information processing (e.g., transcription, replication, translation) in Archaea are simplified versions of their eukaryotic counterparts. This is clearly noticeable in the conservation of sequence and structure of corresponding enzymes (see for example the archaeal DNA-directed RNA polymerase (RNAP)). In Eukarya, post-translational modifications (PTMs) often serve as functional regulatory factors for various enzymes and complexes. Among the various PTMs, methylation and acetylation have been recently attracting most attention. Nevertheless, little is known about such PTMs in Archaea, and cross-methodological studies are scarce. We examined methylation and N-terminal acetylation of endogenously purified crenarchaeal RNA polymerase from Sulfolobus shibatae (Ssh) and Sulfolobus acidocaldarius (Sac). In-gel and in-solution protein digestion methods were combined with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) mass spectrometry analysis. Overall, 20 and 26 methyl-lysines for S. shibatae and S. acidocaldarius were identified, respectively. Furthermore, two N-terminal acetylation sites for each of these organisms were assessed. As a result, we generated a high-confidence data set for the mapping of methylation and acetylation sites in both Sulfolobus species, allowing comparisons with the data previously obtained for RNAP from Sulfolobus solfataricus (Sso). We confirmed that all observed methyl-lysines are on the surface of the RNAP.
authors.affiliation: Proteomics Platform, CIC bioGUNE, ProteoRed-ISCIII, CIBERehd, 48160 Derio, Spain.
authors.forename: Mikel
authors.initials: M
authors.lastname: Azkargorta
authors.forename: Magdalena N
authors.initials: MN
authors.lastname: Wojtas
authors.forename: Nicola G A
authors.initials: NG
authors.lastname: Abrescia
authors.forename: Felix
authors.initials: F
authors.lastname: Elortza
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002853:Chromatography, Liquid; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008745:Methylation; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D016944:Sulfolobus / Q000201:enzymology*; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D021122:Protein Subunits; D012321:DNA-Directed RNA Polymerases; D008239:Lysine