Differential Contribution of Protein Factors and 70S Ribosome to Elongation.

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.

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.