The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.Ribosomes contain three tRNA binding sites, the A, P, and E site, viz. the A site where the decoding takes place, the P site, where the peptidyl-tRNA is located before peptide bond formation, and the E site, which is specific for deacylated tRNA (1-6). During elongation of the nascent peptide chain, each tRNA passes through the ribosomal binding sites in the sequence A 3 P 3 E. To elongate the nascent peptide chain by one amino acid, the ribosome goes through a cycle of reactions, the socalled elongation cycle. The three basic reactions of an elongation cycle are 1) occupation of the A site by an aminoacyl-tRNA according to the corresponding codon at the A site, 2) peptidebond formation, which transfers the already synthesized peptidyl residue to the aminoacyl-tRNA so that the resulting peptidyl-tRNA, prolonged by an amino acid, now resides at the A site, and 3) the translocation reaction, which moves the peptidyl-tRNA to the P site and the deacylated tRNA to the E site.Cross-linking and footprinting studies have identified components of the ribosome that interact with the tRNAs in the specific sites (reviewed in Refs. 7 and 8). Recently, a technique developed by Eckstein and co-workers (9) has been used to investigate the interactions of tRNAs with ribosomal binding sites (10). This method exploits the fact that the addition of iodine (I 2 ) causes a breakage of the sugar-phosphate backbone of phosphorothioated RNA (here tRNA). The cleavage works equally well with phosphates in single or double strands. However, if tight contacts of a distinct phosphate group of a thioated tRNA with a synthetase or a ribosomal component prevents the access of iodine, the phosphate group under observation is protected, and a cleavage is not observed at this position. Highly differentiated and distinct cleavage patterns were found for A and P site-bound tRNAs in the pre-translocational (PRE) 1 state (10). Here we show that the protection patterns of both tRNAs at the A and P sites in the PRE state hardly change when translocated to P and E sites, respectively. Because probably most of the protection patterns are caused by interactions of the tRNA with the respective ribosomal binding sites, the data suggest the existence of a movable ribosomal domain that binds tightly the two ...