Genetic analysis of the E site during RF2 programmed frameshifting

Abstract

The roles of the ribosomal E site are not fully understood. Prior evidence suggests that deacyl-tRNA in the E site can prevent frameshifting. We hypothesized that if the E-site codon must dissociate from its tRNA to allow for frameshifting, then weak codon:anticodon duplexes should allow for greater frameshifting than stronger duplexes. Using the well-characterized Escherichia coli RF2 (prfB) programmed frameshift to study frameshifting, we mutagenized the E-site triplet to all Unn and Cnn codons. Those variants should represent a very wide range of duplex stability. Duplex stability was estimated using two different methods. Frameshifting is inversely correlated with stability, as estimated by either method. These findings indicate that pairing between the deacyl-tRNA and the E-site codon opposes frameshifting. We discuss the implications of these findings on frame maintenance and on the RF2 programmed frameshift mechanism.

FIGURE 1.

Outline of the RF2 programmed frameshift mechanism. (A) Ribosome is paused during search to fill the A site. As is usual during elongation, the anti-Shine–Dalgarno of 16S rRNA is not engaged with the message. tRNAs occupy the E and P sites. The E site codon is varied to all YNN codons and is shown paired to an RXX anticodon. tRNA^Leu ₂ is paired to CUU in the P site. (B) The Shine–Dalgarno engages, which dissociates the E and P site duplexes. The P site tRNA realigns onto the overlapping UUU triplet. (C) Translation resumes when the A site is filled and the Shine–Dalgarno disengages. See text for further details.

FIGURE 2.

FR for the YNN sense codons in the E site. Unn codons are on the left, and Cnn codons are on the right. The five pairs of codons ending in U or C and read by the same tRNA are darkly shaded. Standard errors of the mean are 10% or less.

FIGURE 3.

Plots of ln(FR) versus estimates of codon:anticodon duplex stability; ln(FR) is plotted versus the first (A) and second (B) methods for estimating stability. (C, D) Plots for the Cnn and Unn subsets with FR plotted versus stabilities estimated with the first method.

FIGURE 4.

Plot of ln(FR) versus estimated stability of the potential rephased duplexes.

FIGURE 5.

Plot of FR versus estimated relative rate of aa-tRNA selection at the A site. Data are from Table 1.