Dataset Information

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

ABSTRACT: Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylates some tRNAs. The growth of cells lacking YgfZ is compromised especially at low temperature. The RimO enzyme is homologous to MiaB and thiomethylates a conserved aspartic acid in ribosomal protein S12. To quantitate thiomethylation by RimO, we developed a bottom-up LC-MS² analysis of total cell extracts. We show here that the in vivo activity of RimO is very low in the absence of YgfZ and independent of growth temperature. We discuss these results in relation to the hypotheses relating to the role of the auxiliary 4Fe-4S cluster in the Radical SAM enzymes that make Carbon-Sulfur bonds.

SUBMITTER: Lund T

PROVIDER: S-EPMC10002905 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Essentiality of the <i>Escherichia coli</i> YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Lund Torben T Kulkova Maria Yohanna MY Jersie-Christensen Rosa R Atlung Tove T

International journal of molecular sciences 20230301 5

Enzymes carrying Iron-Sulfur (Fe-S) clusters perform many important cellular functions and their biogenesis require complex protein machinery. In mitochondria, the IBA57 protein is essential and promotes assembly of [4Fe-4S] clusters and their insertion into acceptor proteins. YgfZ is the bacterial homologue of IBA57 but its precise role in Fe-S cluster metabolism is uncharacterized. YgfZ is needed for activity of the radical S-adenosyl methionine [4Fe-4S] cluster enzyme MiaB which thiomethylate ...[more]

PMID: 36902159

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Project description: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.

Dataset Information

Essentiality of the Escherichia coli YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

Publications

Essentiality of the <i>Escherichia coli</i> YgfZ Protein for the In Vivo Thiomethylation of Ribosomal Protein S12 by the RimO Enzyme.

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