Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu-independent step.


After purification, 1 pmol of stalled ribosomes (programmed ribosomes); stalled ribosomes in complex with tm RNA-Smp B in the presence of kirromycin (preaccommodated state), and stalled 70S ribosomes in complex with tm RNA-Smp B, in the absence of kirromycin (accommodated state) were analyzed by SDS/PAGE.Reprint requests to: Rachel Green, Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA; e-mail: [email protected]; fax: (410) 502-6718.Here we use an in vitro selection approach to isolate t RNA miscoding variants that exhibit a globally perturbed t RNA tertiary structure.Interestingly, the most substantial distortions are positioned in the elbow region of the t RNA that closely approaches helix 69 (H69) of the large ribosomal subunit.The importance of these specific interactions to t RNA selection is underscored by our kinetic analysis of both t RNA and r RNA variants that perturb the integrity of this interaction.