Analysis of Interactions of EF-G and EF-Tu with the Ribosome
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Date
2012-05
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The Ohio State University
Abstract
Guanosine triphosphate hydrolases (GTPases) play important roles in many cellular processes, including communication, division, and transport. Two specific GTPases, Elongation Factor Tu (EF-Tu) and Elongation Factor G (EF-G), catalyze different steps of the elongation phase of translation. Although these are extensively studied GTPases, how EF-Tu and EF-G interact with the ribosome is still not fully understood. In order to investigate the interactions between these elongation factors and the ribosome, I analyzed how mutations in helix 5 (h5) of the 16S rRNA of Escherichia coli affect translocation (EF-G function) and decoding (EF-Tu function). Single-point mutations were made at positions 55, 357, and 367 of 16S rRNA and the corresponding ribosomes were purified using affinity chromatography. To determine the effects of the mutations on translocation, the rate of tRNA-mRNA movement was measured under single-turnover conditions. Mutations in h5 had minimal effects on translocation, suggesting that these nucleotides are not critical for the binding or function of EF-G. To determine the effects of the mutations on decoding, apparent rates of EF-Tu-dependent GTP hydrolysis were determined. Mutations A55C and U367G were found to dramatically reduce the rate of GTP hydrolysis, suggesting a specific defect in EF-Tu function. This work sheds light on the role of specific ribosomal residues in elongation. This information will add to our knowledge of translation at the molecular level, and may lead to useful tools for protein engineering.
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Keywords
Ribosome, Elongation Factor G, Elongation Factor Tu, Translation, helix 5