Ribosomes stall when they encounter the end of m RNA without an in-frame stop codon.In bacteria, these nonstop complexes can be rescued by alternative ribosome-rescue factor A (Arf A).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.Docked atomic coordinates are shown by using the Ribbons and Insight II (Accelrys) programs.Fitting coordinates for EF-Tu•GDPNP (mustard color) were used from Protein Data Bank ID code 1B23; the x-ray crystal structure of TLD-Smp Bs (Protein Data Bank ID code 1P6V) was used in two ways, once for the structure of a tm RNA-Smp B moiety, and once using a single Smp B structure (see text); pink for Smp B-2 on the 30S, and gray for Smp B-1 on the 50S subunit.

We use t RNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-t RNAs occurs in two consecutive steps.Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.We have found that the bacterial ribosome uses two proofreading steps following initial selection of transfer RNAs (t RNAs) to maintain high accuracy of translation of the genetic code.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.Collins English Dictionary - Complete & Unabridged 2012 Digital Edition © William Collins Sons & Co.

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