title: A role for the 30S subunit E site in maintenance of the translational reading frame.
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 beta-hairpin of ribosomal protein S7. This mutation (S7DeltaR77-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 S7DeltaR77-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 S7DeltaR77-Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K (1/2) 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.
authors.affiliation: Ohio State Biochemistry Program, The Ohio State University, Columbus, 43210, USA.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Devaraj
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Shoji
authors.forename: Eric D
authors.initials: ED
authors.lastname: Holbrook
authors.forename: Kurt
authors.initials: K
authors.lastname: Fredrick
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D020653:Peptide Elongation Factor G / Q000378:metabolism; D017433:Protein Structure, Secondary; D021381:Protein Transport; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D020653:Peptide Elongation Factor G