Transfer RNA (tRNA) translocates inside the ribosome during translation. We studied the interaction strengths between the ribosome and tRNA at various stages of translocation. We utilized an optical trap to measure the mechanical force to rupture tRNA from the ribosome. We measured the rupture forces of aminoacyl tRNA or peptidyl tRNA mimic from the ribosome in a prepeptidyl transfer state, the pretranslocational state, and the posttranslocational state. In addition, we measured the interaction strength between the ribosome and aminoacyl-tRNA in presence of viomycin. Based on the interaction strengths between the ribosome and tRNA under these conditions, 1), we concluded that tRNA interaction with the 30S subunit is far more important than the interaction with the 50S subunit in the mechanism of translocation; and 2), we propose a mechanism of translocation where the ribosomal ratchet motion, with the aid of EF-G, drives tRNA translocation.
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