title: Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G.
abstract: The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ, U.K.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Deroo
authors.forename: Suk-Joon
authors.initials: SJ
authors.lastname: Hyung
authors.forename: Julien
authors.initials: J
authors.lastname: Marcoux
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Gordiyenko
authors.forename: Ravi Kiran
authors.initials: RK
authors.lastname: Koripella
authors.forename: Suparna
authors.initials: S
authors.lastname: Sanyal
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D021122:Protein Subunits; D012269:Ribosomal Proteins; C512844:rplL protein, E coli