How the ribosome moves along the mRNA during protein synthesis

The movement of a ribosome along the mRNA was assessed by the following experimental strategy. mRNAs were synthesized which contained a short coding sequence with at least four codons and a 32P label at one end and an oligo(C) sequence at the other end. When these mRNAs were fixed on the ribosome with tRNAs specific for the defined codons, the oligo(C) stretches were partially outside of the ribosome, whereas the labeled ends were inside the ribosome and thus protected. The overhanging oligo(C) regions were trimmed with the cytidyl-specific RNase CL3 identifying the nucleotides of the mRNAs, which emerged from the ribosome. An mRNA enters the ribosome at nucleotide 18 +/- 1, when counting starts at the first nucleotide of the P-site codon, and leaves the ribosome at nucleotide -21 +/- 2. The ribosome does not move at either side upon A-site occupation but does so at both sides simultaneously upon translocation. The results further indicate that most of the mRNA stretches downstream and upstream of the coding region are not required for the translocation reaction. It is therefore likely that the tRNAs are pulling the mRNA through the ribosome via codon-anticodon interactions in the course of translocation.