One key question in protein biosynthesis is how the ribosome couples mRNA and tRNA movements to prevent disruption of weak codon-anticodon interactions and loss of the translational reading frame during translocation. Here we report the complete path of mRNA on the 70S ribosome at the atomic level (3.1-A resolution), and we show that one of the conformational rearrangements that occurs upon transition from initiation to elongation is a narrowing of the downstream mRNA tunnel. This rearrangement triggers formation of a network of interactions between the mRNA downstream of the A-site codon and the elongating ribosome. Our data elucidate the mechanism by which hypermodified nucleoside 2-methylthio-N6 isopentenyl adenosine at position 37 (ms(2)i(6)A37) in tRNA(Phe)(GAA) stabilizes mRNA-tRNA interactions in all three tRNA binding sites. Another network of contacts is formed between this tRNA modification and ribosomal elements surrounding the mRNA E/P kink, resulting in the anchoring of P-site tRNA. These data allow rationalization of how modification deficiencies of ms(2)i(6)A37 in tRNAs may lead to shifts of the translational reading frame.