Protein biosynthesis, also called translation, refers to mRNA-programmed synthesis of a polypeptide on the ribosome. tRNAs are required to move efficiently between several positions on the ribosome during the elongation phase of translation. Although structures of tRNA bound to ribosomes are available, the dynamics and mechanism of tRNA movement and structural change are incompletely understood. The classic L-shape of tRNA undergoes considerable distortion during the elongation cycle, leading to the suggestion that the tRNA tertiary core region, which stabilizes the L-shape, actively interacts with the ribosome. Here, using fluorescence labeling of tRNA and mutagenesis of highly conserved nucleotides within the tertiary core, combined with binding and single turnover rapid kinetics assays, we monitor tRNA movement on the ribosome, examine the importance of the tertiary core region for each step of the elongation cycle, identify interactions and intermediates that are important for the accuracy and efficiency of protein biosynthesis, and determine antibiotic effects on elongation.