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Preaccommodating

preaccommodating-62

First, aa-t RNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-t RNA affinity to EF-Tu.Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu−independent step.

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Arf A binds within the ribosomal m RNA channel and substitutes for the absent stop codon in the A site by specifically recruiting release factor 2 (RF2), initially in a compact pre-accommodated state.-translation is performed by transfer-messenger RNA (tm RNA), a specialized RNA acting as both a t RNA and an m RNA, associated with small protein B (Smp B).So far, a clear view of the structural movements of both the protein and RNA necessary to perform accommodation is still lacking.Quantification was processed by using Image Quant software (Amersham Pharmacia, Piscataway, NJ).Relative occupancy of tm RNA within each of the purified complexes was obtained by using radiolabeled tm RNA (see .Two picomoles of the same ribosomal complexes were analyzed by Western blotting using anti-Smp B antibodies.

Relative amounts of Smp B per ribosome were obtained by comparing the signal intensity with gradually increasing amounts of purified Smp B.

Aminoacyl-t RNAs (aa-t RNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu.

We use t RNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-t RNAs occurs in two consecutive steps.

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.

Docked atomic coordinates are shown by using the Ribbons and Insight II (Accelrys) programs.