A role for the 30S subunit E site in maintenance of the translational reading frame.
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.
Devaraj
Aishwarya
A
Ohio State Biochemistry Program, The Ohio State University, Columbus, 43210, USA.
Shoji
Shinichiro
S
Holbrook
Eric D
ED
Fredrick
Kurt
K
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*
D020653:Peptide Elongation Factor G