Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Skipper, Nigel; Thomas, David Y.; Lau, Peter C. K.

doi:10.1002/j.1460-2075.1984.tb01769.x

Cited by 81 publications

(33 citation statements)

References 35 publications

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“…2B). Hybridization of the labeled products of the M1-H and M1-L VLPs with M13 clones that contained M1 cDNA inserts in both orientations (27) (Table 3) showed that both peaks synthesize predominantly plus-strand (i.e., message strand) RNA. The phenolextracted RNA product is mainly single stranded as judged VOL.…”

Section: Methodsmentioning

confidence: 99%

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Esteban¹,

Wickner²

1986

Mol. Cell. Biol.

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Killer strains of Saccharomyces cerevisiae bear at least two different double-stranded RNAs (dsRNAs) encapsidated in 39-nm viruslike particles (VLPs) of which the major coat protein is coded by the larger RNA (L-A dsRNA). The smaller dsRNA (M1 or M2) encodes an extracellular protein toxin (Kl or K2 toxin). Based on their densities on CsCl gradients, L-A-and Ml-containing particles can be separated. Using this method, we detected a new type of Ml dsRNA-containing VLP (Ml-H VLP, for heavy) that has a higher density than those previously reported (Ml-L VLP, for light). Ml-H and Ml-L VLPs are present together in the same strains and in all those we tested. Ml-H, Ml-L, and L-A VLPs all have the same types of proteins in the same approximate proportions, but whereas L-A VLPs and Ml-L VLPs have one dsRNA molecule per particle, MI-H VLPs contain two Ml dsRNA molecules per particle. Their RNA polymerase produces mainly plus single strands that are all extruded in the case of M1-H particles but are partially retained inside the Ml-L particles to be used later for dsRNA synthesis. We show that MI-H VLPs are formed in vitro from the M1-L VLPs. We also show that the peak of Ml dsRNA synthesis is in fractions lighter than Ml-L VLPs, presumably those carrying only a single plus M1 strand. We suggest that VLPs carrying two M1 dsRNAs (each 1.8 kilobases) can exist because the particle is designed to carry one L-A dsRNA (4.5 kilobases).

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Section: Methodsmentioning

confidence: 99%

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Esteban¹,

Wickner²

1986

Mol. Cell. Biol.

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“…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.…”

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confidence: 99%

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confidence: 99%

Kinetics of Ribosomal Pausing during Programmed −1 Translational Frameshifting

Lopinski

Dinman

Bruenn

2000

Molecular and Cellular Biology

108

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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...

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“…In S. cerevisiae, the kl toxin consists of two subunits, alpha (103 amino acids) and beta (83 amino acids), joined by two disulfide bonds (5,42,54). These are encoded on one double-stranded RNA (dsRNA) of the S. cerev'isiae virus (ScV) and synthesized as a prepropolypeptide of 317 amino acids, which is cleaved by signal peptidase, the KEXi and KEX2 proteases, and by at least one more unspecified protease during its maturation (5,9,15,17,22,42,54 P6, respectively (24, 26-30). Their mode of action is unknown, but like the S. cerevisiae kl toxin, the KP6 toxin has cell wall receptors (44).…”

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confidence: 99%

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

Tao¹,

Ginsberg²,

Banerjee³

et al. 1990

Mol. Cell. Biol.

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There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae kl killer toxin, also encoded by a double-stranded RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a threeamino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.Killer toxins are known in eight genera of yeasts (53).Those of Saccharomyces cerevisiae, Picahia kluyveri, and Kluyv'eromyces lactis are best characterized (12,23,46). In S. cerevisiae, the kl toxin consists of two subunits, alpha (103 amino acids) and beta (83 amino acids), joined by two disulfide bonds (5,42,54). These are encoded on one double-stranded RNA (dsRNA) of the S. cerev'isiae virus (ScV) and synthesized as a prepropolypeptide of 317 amino acids, which is cleaved by signal peptidase, the KEXi and KEX2 proteases, and by at least one more unspecified protease during its maturation (5,9,15,17,22,42,54 P6, respectively (24, 26-30). Their mode of action is unknown, but like the S. cerevisiae kl toxin, the KP6 toxin has cell wall receptors (44). Unlike the ScV kl toxin, in which the two subunits are joined by disulfide bonds (5), or the K. lac tis toxin, in which the three subunits are present in a (probably) noncovalently bonded complex (43), the KP6 toxin polypeptides interact with the cell independently, as monomers (37). Genetic and in vitro translation data did not indicate conclusively whether the two KP6 polypeptides were synthesized as one preprotoxin or as two (28,37).We have shown that the KP6 toxin polypeptides are synthesized as one preprotoxin of 219 amino acids with a typical signal sequence. The preprotoxin is cleaved by an enzyme with specificity similar to that of the S. cerevisiae KEX2 enzyme (22) and by at least one other enzyme with specificity like that of the enzyme which cleaves off the N terminus of the S. cerevisiae kl preprotoxin. The result is two secreted polypeptides alpha (78 amino acids) and beta (81 Amino acids), whose C termini are predicted in analogy with the kl toxin. The KP6 preprotoxin was properly processed in S. cerev-isiae, and active KP6 alpha and beta were secreted. KP6 alp...

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Cloning and sequencing of the preprotoxin-coding region of the yeast M1 double-stranded RNA.

Cited by 81 publications

References 35 publications

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Three different M1 RNA-containing viruslike particle types in Saccharomyces cerevisiae: in vitro M1 double-stranded RNA synthesis.

Kinetics of Ribosomal Pausing during Programmed −1 Translational Frameshifting

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

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