1. A role for the 30S subunit E site in maintenance of the translational reading frame
- Author
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Kurt Fredrick, Eric D. Holbrook, Aishwarya Devaraj, and Shinichiro Shoji
- Subjects
Genetics ,Translational frameshift ,Molecular Sequence Data ,Frameshifting, Ribosomal ,E-site ,Ribosome Subunits, Small, Bacterial ,Gene Expression Regulation, Bacterial ,Biology ,Peptide Elongation Factor G ,Ribosome ,Article ,Protein Structure, Secondary ,Frameshift mutation ,Open Reading Frames ,Protein Transport ,Open reading frame ,Ribosomal protein ,Mutation ,Transfer RNA ,Escherichia coli ,Amino Acid Sequence ,Molecular Biology ,50S - Abstract
The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the β-hairpin of ribosomal protein S7. This mutation (S7ΔR77–Y84) increases both −1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7ΔR77–Y84 also stimulates +1 programmed frameshifting during prfB′-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7ΔR77–Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K1/2 for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.
- Published
- 2008
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