Distance moved by transfer RNA during translocation from the A site to the P site on the ribosome

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Singlet-singlet energy transfer has been used to measure the distance between the midsections of two transfer RNAs bound to the same ribosome. Two Escherichia coli tRNA species were modified by the covalent attachment of a fluorescent dye to 4-thiouridine: tRNA f Met was reacted with IAEDANS ‡ ‡ Abbreviation used: IAEDANS, 5-[2-(2-iodoacetamido)ethylamino]-l-naphthalenesulfonic acid; s 4U, 4-thiouridine; Ψ, pseudouridine; IAAF, 5-iodoacetamidofluorescein; s 4U-AEDANS, adduct of s 4U base and IAEDANS; s 4U-F, adduct of s 4U base and IAAF; tRNA f Met-AEDANS 8, adduct between IAEDANS and s 4U 8 base of tHNA f Met; tRNA Phe-F 8, adduct between IAAF and s 4U 8 base of tRNA Phe; EF-Tu, elongation factor Tu; EF-G, elongation factor G. to give tRNA f Met-AEDANS 8, and tRNA Phe with 5-iodoacetamidofluorescein to give tRNA Phe-F 8. These fluorescent-labeled tRNAs were purified by chromatography on RPC-5. The modified tRNA f Met-AEDANS 8 interacted to the same extent as the unmodified tRNA with methionyl-tRNA synthetase, transformylase, initiation factors, ribosomes and the peptidyltransferase. The ability of tRNA Phe-F 8 to interact with phenylalanyl-tRNA synthetase, EF-Tu · GTP and the ribosomal complex was only slightly diminished by the presence of the fluorescein dye. Ribosomal complexes were prepared for the energy transfer experiments by binding fMet-tRNA f Met-AEDANS 8 (the donor) to the P site using an A-U-G-U 4–5 message, and then binding Phe-tRNA Phe-F 8 (the acceptor) to the A site using EF-Tu · GTP. The necessary control samples were prepared in parallel using unmodified tRNAs in place of the donor, the acceptor, or both. The fluorescence of the donor dye in the P site was strongly quenched (~ 70%) when the acceptor dye was present in the A site. This high efficiency of energy transfer corresponds to a separation distance of 26(±4)Å between the dyes. The midsections of the tRNAs bound in the A and P sites are therefore located close to each other, separated by only 2 to 10 Å, and translocation from the A site into the immediately adjacent P site requires a movement of less than 30 Å. This arrangement of the tRNAs suggests that the ejection of the deacylated tRNA from the P site might be powered by the electrostatic repulsion between the tRNAs bound in the A and P sites.