title: A coordinated proteomic approach for identifying proteins that interact with the E. coli ribosomal protein S12.
abstract: The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the 30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader , M. B. ; Costantino , N. ; Elkins , C. A. ; Chen , C. Y. ; Patel , I. ; Makusky , A. J. ; Choy , J. S. ; Court , D. L. ; Markey , S. P. ; Kowalak , J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011 , 10 , M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography-tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.
authors.affiliation: Laboratory of Neurotoxicology, National Institute of Mental Health , Bethesda, Maryland 20892, United States.
authors.forename: Michael Brad
authors.initials: MB
authors.lastname: Strader
authors.forename: William Judson
authors.initials: WJ
authors.lastname: Hervey
authors.forename: Nina
authors.initials: N
authors.lastname: Costantino
authors.forename: Suwako
authors.initials: S
authors.lastname: Fujigaki
authors.forename: Cai Yun
authors.initials: CY
authors.lastname: Chen
authors.forename: Ayca
authors.initials: A
authors.lastname: Akal-Strader
authors.forename: Chibueze A
authors.initials: CA
authors.lastname: Ihunnah
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Makusky
authors.forename: Donald L
authors.initials: DL
authors.lastname: Court
authors.forename: Sanford P
authors.initials: SP
authors.lastname: Markey
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Kowalak
mesh_terms: D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D040901:Proteomics; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D029968:Escherichia coli Proteins; D012269:Ribosomal Proteins; C034212:ribosomal protein S12