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. 2007 Apr;13(4):606-13.
doi: 10.1261/rna.412707. Epub 2007 Feb 28.

An mRNA sequence derived from the yeast EST3 gene stimulates programmed +1 translational frameshifting

Dwayne Taliaferro  1 , Philip J Farabaugh
Affiliations

An mRNA sequence derived from the yeast EST3 gene stimulates programmed +1 translational frameshifting

Dwayne Taliaferro et al. RNA. 2007 Apr.

Abstract

Programmed translational frameshift sites are sequences in mRNAs that promote frequent stochastic changes in translational reading frame allowing expression of alternative forms of protein products. The EST3 gene of Saccharomyces cerevisiae, encoding a subunit of telomerase, uses a programmed +1 frameshift site in its expression. We show that the site is complex, consisting of a heptameric sequence at which the frameshift occurs and a downstream 27-nucleotide stimulator sequence that increases frameshifting eightfold. The stimulator appears to be modular, composed of at least three separable domains. It increases frameshifting only when ribosomes pause at the frameshift site because of a limiting supply of a cognate aminoacyl-tRNA and not when pausing occurs at a nonsense codon. These data suggest that the EST3 stimulator may modulate access by aminoacyl-tRNAs to the ribosomal A site by interacting with several targets in a ribosome paused during elongation.

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Figures

FIGURE 1.
FIGURE 1.
Structure of the EST3 gene and frameshift region. The diagram at the bottom shows the arrangement of the two open reading frames and the frameshift site (FS) within the overlap region. Above is the DNA sequence of the heptameric frameshift site (white letters on black) and the 30 nt both upstream and downstream. Below the DNA sequence is the encoded protein product in the zero (above) and +1 (below) reading frames with the amino acids encoded into the frameshift product in black and the arrow indicating decoding by frameshifting. The extent of three oligonucleotides referred to in Figure 2 (EST3 67 mer, 5′ 37 mer, and 3′ 37 mer are indicated above the DNA sequence.
FIGURE 2.
FIGURE 2.
Identifying the EST3 frameshift stimulator. Shown is the frameshift efficiency in percent (±SEM expressed as a percent) for the indicated constructs derived from the EST3 gene, described in the text.
FIGURE 3.
FIGURE 3.
Deletion mapping of the EST3 stimulator. Each bar represents the frameshifting efficiency, as in Figure 2, for one of a series of reporters that includes increasing amounts of the region downstream of the EST3 frameshift site.
FIGURE 4.
FIGURE 4.
Missense mutagenesis of the EST3 stimulator. Each bar represents β-galactosidase activity (±SEM) expressed from a reporter involving a wild-type (Wt) or missense mutant EST3 stimulator. Each wild-type nucleotide appears below the graph with a subscript indicating the position; the identity of each tested mutation appears just above in groups of 3. Black bars represent results judged by T-test as different from wild type (P ≤ 0.005). Asterisks indicate mutations that introduce an in frame nonsense codon, which strongly reduces expression of lacZ.
FIGURE 5.
FIGURE 5.
Alignment of budding yeast EST3 stimulators. The sequence of the frameshift sites and downstream stimulator sequences from 10 budding yeast species. Identity is indicated by shading.
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