Kinetics of Ribosomal Pausing during Programmed −1 Translational Frameshifting

Abstract

In the Saccharomyces cerevisiae double-stranded RNA virus, programmed ؊1 ribosomal frameshifting is responsible for translation of the second open reading frame of the essential viral RNA. A typical slippery site and downstream pseudoknot are necessary for this frameshifting event, and previous work has demonstrated that ribosomes pause over the slippery site. The translational intermediate associated with a ribosome paused at this position is detected, and, using in vitro translation and quantitative heelprinting, the rates of synthesis, the ribosomal pause time, the proportion of ribosomes paused at the slippery site, and the fraction of paused ribosomes that frameshift are estimated. About 10% of ribosomes pause at the slippery site in vitro, and some 60% of these continue in the ؊1 frame. Ribosomes that continue in the ؊1 frame pause about 10 times longer than it takes to complete a peptide bond in vitro. Altering the rate of translational initiation alters the rate of frameshifting in vivo. Our in vitro and in vivo experiments can best be interpreted to mean that there are three methods by which ribosomes pass the frameshift site, only one of which results in frameshifting.A 4.6-kb double-stranded RNA (dsRNA) virus, ScV-L-A (also known as ScV-L1), can be found in many laboratory strains of the yeast Saccharomyces cerevisiae. Like most fungal viruses, ScV-L-A is exclusively cytoplasmic, has no infectious cycle, and is known to be naturally transmitted only by budding or by cell fusion during mating (16). It is a member of the Totiviridae, which are dsRNA viruses with a single essential viral dsRNA (6).The viral genome has two open reading frames (ORFs) on its plus strand that overlap by 129 bases (Fig. 1). The first ORF is designated cap or gag (after the retrovirus nomenclature) and encodes the 76-kDa major capsid polypeptide. The second, pol, encodes the viral RNA-dependent RNA polymerase (12,14,21). Translation of pol as a 171-kDa Cap-Pol fusion protein is permitted by a programmed Ϫ1 ribosomal frameshift (12,14,21,40) (Fig. 1). A second, separately encapsidated viral dsRNA (M 1 ) encodes a secreted polypeptide toxin (k1 killer toxin) that provides a convenient phenotype for detecting synthesis of 34,37).Programmed, or directed, ribosomal frameshifting (PRF) is of particular value to viruses that package their plus strands, as it eliminates the need to splice their mRNAs and reduces the risk of packaging defective genomes. It also regulates the ratio of viral proteins synthesized. Examples of the phenomenon (both ϩ1 and Ϫ1 shifts) have been found in a wide range of systems. In addition to the ScV systems, programmed translational frameshifting has been identified in retroviruses (17, 23, 30, 32); coronaviruses (5, 20); giardiaviruses, which are also members of the Totiviridae (41); two bacterial genes (3, 8); bacteriophage genes (7); astroviruses (28); the yeast EST3 gene (27); and the rat, mouse, Xenopus, and Drosophila ornithine decarboxylase antizymes (29) (reviewed in references 13 and 18). A signifi...

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…In the context of frameshifting events, it has been determined that pausing is necessary but not sufficient to induce -1 frameshifting events (110). These studies suggest the pseudoknot functions as a kinetic barrier to force-induced unfolding, essentially placing the unfolding of the entire pseudoknot under kinetic control (5).…”

“…In addition to the frameshifting process being kinetically controlled from the rate of unfolding, a model has been proposed that suggests that the rate of refolding will affect frameshifting efficiency (110). In short, the number of translocating ribosomes on a single mRNA molecule might also have a detectable influence on ensemble average frameshifting efficiency.…”

“…In short, the number of translocating ribosomes on a single mRNA molecule might also have a detectable influence on ensemble average frameshifting efficiency. In this model, the rate of refolding the pseudoknot after unfolding and decoding by the first ribosome may have a significant impact on overall frameshift efficiency because a lagging ribosome(s) may encounter a stimulatory element that remains unfolded (110).…”

Efficient stimulation of site-specific ribosome frameshifting by antisense oligonucleotides

“…Although the mechanism of pseudoknot stimulation is unknown, the IBV (Somogyi et al 1993;Kontos et al 2001) and Saccharomyces cerevisiae L-A virus (Tu et al 1992;Lopinski et al 2000) pseudoknots have been shown to pause the ribosome. Pausing is very likely the mechanism of frameshift stimulation in examples where frameshifting is stimulated by a stop codon (Weiss et al 1987(Weiss et al , 1990Gramstat et al 1994) or a "hungry" codon in the A-site due to limitation in the abundance of aminoacyl tRNA (for review, see Gallant and Lindsley 1998).…”

The 9-Å solution: How mRNA pseudoknots promote efficient programmed −1 ribosomal frameshifting

“…Modeling of the MMTV frameshift signal into the Escherichia coli ribosome by Giedroc et al (2000) supports the notion that the linker region positions the pseudoknot on the surface of the ribosome when the A-and P-sites are occupied by the slippery site sequence. The func-tion of the downstream secondary structure is to induce elongating ribosomes to pause (Rice et al 1985), and its ability to induce ribosomal pausing is critical for efficient −1 PRF to occur (Tu et al 1992;Somogyi et al 1993;Lopinski et al 2000;Kontos et al 2001). The generally accepted mechanism of −1 PRF is as follows: (1) The secondary mRNA structure forces elongating ribosomes to pause, and the length of the linker is such that the ribosomal A-and P-site bound aminoacyl-(aa-) and peptidyl-tRNAs are positioned over the slippery site; (2) the sequence of the slippery site allows for re-pairing of the tRNAs to the −1 frame codons after they "simultaneously slip" by one base in the 5Ј direction along the mRNA; and (3) subsequent melting of the downstream mRNA secondary structure allows the ribosome to continue elongation of the nascent polypeptide in the new translational reading frame.…”