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Abstract
The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.
Highlights
The ribosome is a macromolecular complex that translates the genetic information into the amino acid sequence of proteins
The most of genetic and biochemical data revealing the peculiarities of the functioning of translation refer to mesothermal microorganism Escherichia coli (E. coli), while structural interpretation of protein biosynthesis primarily relates to the data obtained using components of thermophilic microorganisms, in particular, Thermus thermophilus (T. thermophilus)
Kinetics of tRNA Interaction with the Ribosome Is Determined by the Properties of Elongation Factor EF-Tu
Summary
The ribosome is a macromolecular complex that translates the genetic information into the amino acid sequence of proteins. E. coli reconstituted in vitro translation system with the elongation factors substituted for those from the thermophilic microorganism T. thermophilus was used to study two fundamental reactions of the elongation cycle: the A-site binding of aa-tRNA and the displacement of peptidyl-tRNA from the A to the peptidyl (P) site of the ribosome during translocation. This approach provides a biochemical characterisation of individual thermophilic components in the context of certain partial translation reactions. It provides a deeper understanding of the mesophilic system’s functioning and merges extensive structural information to biochemical and genetic data
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