Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G

• Published in: ACS chemical biology Vol. 7; no. 6; pp. 1120 - 1127
• Main Authors: Deroo, Stéphanie, Hyung, Suk-Joon, Marcoux, Julien, Gordiyenko, Yuliya, Koripella, Ravi Kiran, Sanyal, Suparna, Robinson, Carol V
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.06.2012
• Subjects: Biochemistry, Molecular Biology; Escherichia coli - chemistry; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Kinetics; Life Sciences; Mass Spectrometry; Peptide Elongation Factor G - metabolism; Protein Binding; Protein Multimerization; Protein Subunits - chemistry; Protein Subunits - metabolism; Ribosomal Proteins - chemistry; Ribosomal Proteins - metabolism; Ribosomes - chemistry; Ribosomes - metabolism; Structural Biology
• ISSN: 1554-8929; 1554-8937; 1554-8937
• DOI: 10.1021/cb300081s

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.