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 b-hairpin of ribosomal protein S7. This mutation (S7DR77-Y84) increases both À1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7DR77-Y84 also stimulates +1 programmed frameshifting during prfB9-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 S7DR77-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.

Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

“…E-site dissociation rates and partitioning between the POST pathways over a range of TC concentrations show that ribosomes on the 2-3-2 pathway effectively trap the deacylated tRNA in the E site until the A site becomes occupied. The tRNA occupancy of the E site inhibits EF-G assisted translocation (shown above) and has been implicated in preventing amino acid misincorporation and random frame-shifting (21,48,49), as well as in assisting programmed frame-shifting (23,24). Future single molecule studies will provide direct tests of the functional effects of E-site binding, and how A-site binding modulates these effects, on these and other processes [e.g., binding of accessory protein factors (50)(51)(52)(53)(54), entry of the nascent peptide chain into the exit tunnel (46) and formation of the Shine-Dalgarno helix (46,55)].…”

Ribosomal Protein S12 and Aminoglycoside Antibiotics Modulate A-site mRNA Cleavage and Transfer-Messenger RNA Activity in Escherichia coli

“…Plasmids pKW1, pKW11, pKW23, pPW500, pCH201, and pAD8 have been described previously (8,(22)(23)(24). DNA fragments encoding the trc promoter and the N-terminal domain of phage cI repressor (N) were PCR-amplified from plasmid pPW500 using oligonucleotide primers containing restriction sites (underlined bases).…”

“…Dual-Luciferase Assay-E. coli strains carrying plasmid pAD8 (24) were grown overnight in LB medium supplemented with 150 g/ml ampicillin. The following day, cells were diluted 1:200 into fresh LB containing 150 g/ml ampicillin and 50 M streptomycin as indicated.…”

“…Therefore, we assessed miscoding in several of the rpsL mutants to determine whether a correlation exists between error restriction and reduced tmRNA⅐ SmpB activity. Stop codon read-through was measured using a Renilla luciferase-firefly luciferase fusion construct, in which the firefly luc gene contains an in-frame UGA stop codon at position 417 (24). This construct only produces firefly luciferase activity when the UGA codon is inappropriately decoded as a sense codon.…”

Intersubunit Bridges of the Bacterial Ribosome

“…In line with this idea, in the cryo-EM reconstruction of the E. coli ribosome with E-site tRNA, there is clear evidence for bridge interaction between L2 (Gln162, Arg174, Arg176, Met200, Arg268) and h23/h24 backbone at similar region as seen in T. thermophilus ribosome (Figure 6D, Table 2). The dynamics at B7b in response to E site occupancy might contribute to the reported effects of E-site tRNA on decoding 69 , translocation 70 , and frame maintenance 71; 72 .…”