Comparative study of the effects of heptameric slippery site composition on -1 frameshifting among different eukaryotic systems

Abstract

Studies of programmed -1 ribosomal frameshifting (-1 PRF) have been approached over the past two decades by many different laboratories using a diverse array of virus-derived frameshift signals in translational assay systems derived from a variety of sources. Though it is generally acknowledged that both absolute and relative -1 PRF efficiency can vary in an assay system-dependent manner, no methodical study of this phenomenon has been undertaken. To address this issue, a series of slippery site mutants of the SARS-associated coronavirus frameshift signal were systematically assayed in four different eukaryotic translational systems. HIV-1 promoted frameshifting was also compared between Escherichia coli and a human T-cell line expression systems. The results of these analyses highlight different aspects of each system, suggesting in general that (1) differences can be due to the assay systems themselves; (2) phylogenetic differences in ribosome structure can affect frameshifting efficiency; and (3) care must be taken to employ the closest phylogenetic match between a specific -1 PRF signal and the choice of translational assay system.

FIGURE 1.

Frameshifting efficiency from individual slippery sites varies depending on the translational assay system. Luminescence from test constructs is expressed as a percentage of the nonframe-shifting control for different slippery sites. The shading of the bars represents frameshifting stimulated by different translational systems: dark gray for Vero cells, light gray for reticulocyte lysate, white for yeast cells, and black for wheat germ lysate. Percentages of frameshifting and standard errors are indicated in both the figure and the table below.

FIGURE 2.

Influence of the A- and the P-site codons on −1 frame-shifting in different translational systems. (A) Influence of the A-site codon. (B) Influence of the P-site codon. Frameshifting efficiencies were determined and expressed as fold change of the wild-type sequence (U UUA AAC) for each construct. The shading of the bars is the same as in Figure 1 ▶. P-values for fold change are in Table 1 ▶.

FIGURE 3.

Effects of the seventh position of the slippery site on −1 frameshifting in different translational systems. (A) Effects with a SARS-like slippery site. (B) Effects with a (L–A)-like slippery site. Frameshifting efficiencies were determined and expressed as fold change of the SARS wild-type sequence (U UUA AAC) for each construct. The shading of the bars is the same as in Figure 1 ▶. P-values for fold change are in Table 1 ▶.