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

1982; Elsevier BV; Volume: 156; Issue: 1 Linguagem: Inglês

10.1016/0022-2836(82)90462-4

1089-8638

Arthur E. Johnson, Harvey J. Adkins, Elizabeth Matthews, Charles R. Cantor,

RNA Research and Splicing

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: tRNAfMet was reacted with IAEDANS‡ to give tRNAfMet-AEDANS8, and tRNAPhe with 5-iodoacetamidofluorescein to give tRNAPhe-F8. These fluorescent-labeled tRNAs were purified by chromatography on RPC-5. The modified tRNAfMet-AEDANS8 interacted to the same extent as the unmodified tRNA with methionyl-tRNA synthetase, transformylase, initiation factors, ribosomes and the peptidyltransferase. The ability of tRNAPhe-F8 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-tRNAfMet-AEDANS8 (the donor) to the P site using an A-U-G-U4–5 message, and then binding Phe-tRNAPhe-F8 (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.