Volume 270, Number 18, Issue of May 5, pp. 10361-10364, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc. 

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Philip J. Farabaugh (§)

From the (1) Department of Biological Sciences, University of Maryland, Baltimore, Maryland 21228 

ABSTRACT

INTRODUCTION

Structure of Ty Elements

Programmed Translational Frameshifting in Ty Elements

Relevance of Frameshifting to the Ty Life Cycle

Proteolytic Processing and Morphogenesis of the Virus-like Particle

Summary and Perspectives

FOOTNOTES

ACKNOWLEDGEMENTS

REFERENCES

INTRODUCTION 

Transposable elements in the yeast Saccharomyces cerevisiae consist of a family of retrotransposons, Ty 1(1) , Ty 2(2) , Ty 3(3) , Ty 4(4, 5) , and Ty 5(6) .() Ty elements transpose through an RNA intermediate using the same replication and integration strategy employed by the metazoan retroviruses (7, 8, 9) . Because of this similarity, they provide an attractive model for virus-host interaction in a genetically tractable eukaryote. Much of the work on Ty elements has therefore focused on the transposition cycle and its regulation. Since the Ty mRNA has two roles, as messenger for Ty-encoded proteins and as substrate for reverse transcription, alterations in the level of Ty mRNA can drastically affect the efficiency of transposition (10) . However, much of the regulation of transposition occurs post-transcriptionally, including translational control (programmed translational frameshifting (11, 12) ) and post-translational control (proteolytic processing (13, 14, 15, 16) ). This review will focus on recent advances in our understanding of post-transcriptional regulation of transposition of Ty elements.

Structure of Ty Elements 

Ty elements are composites of an approximately 5-kilobase central region flanked by long terminal repeats, LTRs() (Fig. 1 ) (for a review see Refs. 17-19). Transcripts of the Ty 1, Ty 2, and Ty 3 initiate in the 5`-LTR and terminate in the 3`-LTR, generating an RNA with a short terminal duplication, a structure essential to the process of reverse transcription. Like metazoan retroviruses, they express one of their primary translation products by an unusual mechanism. Ty elements include two open reading frames, TYA and TYB, which are analogous to the retroviral gag and pol genes, respectively.() As expected for a eukaryotic mRNA, the first potential initiator codon in the mRNAs of Ty elements is the AUG at the 5`-end of the TYA gene. One would predict that this RNA would express only the TYA protein. This leaves open the question of how the TYB gene is expressed since there is no subgenomic RNA that could be normally translated to express TYB. In the mid-1980s it became clear that in Ty elements (20, 21, 22) , as in certain retroviruses (23) , expression of the downstream TYB gene occurred by translational frameshifting. The mechanism used by Ty elements had to be different, since in retroviruses the pol gene overlaps the end of gag in the -1 frame, while in Ty elements TYB overlaps the 3`-end of TYA in the +1 frame. This difference in sign in turn reflects a profound difference in mechanism between the two (reviewed in Refs. 24 and 25).

Programmed Translational Frameshifting in Ty Elements 

Ty elements encode a protein of the size predicted for the TYA product and a second product of the size predicted for a TYA- TYB fusion protein (20, 21, 26, 27) . Expression of the putative fusion protein as expected required continued translation through TYA(20) . RNA mapping studies eliminated the hypothesis that expression of TYB occurred by RNA splicing (20, 21) . Another hypothesis, that the editing of the transcript by eliminating one nucleotide might fuse the TYA and TYB reading frames, was definitively eliminated by cDNA sequencing of the mRNA, which showed that the mRNA was colinear with the DNA template (28) .

Frameshifting Induced by tRNA Slippage

Unconventional Frameshifting without tRNA Slippage

Relevance of Frameshifting to the Ty Life Cycle 

What function does translational frameshifting serve for Ty elements? Clearly, the near ubiquity among retroviruses and retrotransposons of alternative translational events in generating Gag-Pol fusion proteins (reviewed in Refs. 31 and 32) speaks to the importance of this event in the life cycle of the elements. One purpose of this mechanism is that it allows for regulation of the stoichiometry of the structural ( i.e. Gag) and enzymatic (Pol) products, which appears crucial for efficient reverse transcription (33) .

Since reverse transcription occurs within gag-encoded viral core particles, the gag portion of a Gag-Pol fusion protein targets it to assemble as part of that core, placing the enzymatic activities within the forming particle (reviewed in Ref. 34). The second purpose of frameshifting relates to the fact that a packaging signal, termed , is present within the gag gene, which directs insertion of the mRNA into the forming viral core (35) . If the Gag-Pol fusion were made by some pretranslational mechanism, an mRNA would be produced in which the two genes would be in-frame and which could be packaged into core particles (36) . An element in which the gag and pol genes are fused is not capable of futher replication (37, 38, 39, 40) . Thus, the putative gag- pol splice would lead to generation of defective particles. Thus frameshifting provides a morphogenetic tool while avoiding a potential genetic problem.

The fact that frameshifting is crucial to the life cycle of the element is demonstrated by the fact that altering the efficiency of frameshifting can interfere with transposition. Xu and Boeke (37) demonstrated that overproducing the AGG-decoding tRNA caused a drastic decrease in transposition. Kawakami et al.(41) later identified a strain deleted for the only gene encoding tRNA, termed HSX1. Surprisingly, the  hsx1 strain is viable, presumably because AGG continues to be decoded by the near-cognate tRNA specific for AGA. However, in the  hsx1 strain frameshifting at the Ty 1 site was dramatically increased, and concomitantly, transposition of Ty 1 was drastically reduced (42) . The defect appears to be at the level of proteolytic processing (42) , as described below in detail. Thus, changes either increasing or decreasing the efficiency of frameshifting can have a profound effect of transposition of the element by altering the very sensitive stoichiometry of Ty 1 gene expression.

Proteolytic Processing and Morphogenesis of the Virus-like Particle 

Ty elements encode two primary translation products (except Ty 5), both of which are proteolytically processed during formation of mature virus-like particles (VLPs). A protease (PR), encoded as part of the TYB gene, is responsible for this proteolysis. Processing has been studied in both Ty 1 and Ty 3 elements, with similar results (see Fig. 3 ). Early work on Ty 1 identified three predominant protein products, termed p1 (58 kDa), p2 (54 kDa), and p3 (190 kDa) (13, 26, 28, 43) . Two of the proteins are primary products, p1 from the TYA gene and p3 as a fusion product jointly encoded by TYA and TYB. The third, p2, is a processed TYA product generated when PR removes an extreme C-terminal oligopeptide (13, 15) . The p2 protein is the major protein constituent of the VLPs (13, 15) , the probable capsid protein (CA). PR also processes the TYA- TYB polyprotein of Ty 1, releasing three polypeptides: reverse transcriptase/RNase H (RT/RH, 60 kDa), integrase (IN, 90 kDa), and PR itself (23 kDa) (14, 44) .

Blocking Processing Interferes with Transposition

The defect of the PR-mutant VLPs appears to be that cDNA synthesis is much reduced. Mutant Ty 3 particles had background levels of reverse transcriptase, suggesting that processing is essential for enzyme activity (46) . This was, however, not true for the unprocessed Ty 1 polyprotein. Though reverse transcriptase was present, endogenous cDNA synthesis was reduced to background levels (16) . The VLPs contain about 10-fold less RNA, either because of defective packaging or degradation, though it is not clear how a 10-fold reduction could eliminate transposition. Perhaps the VLPs also lack other required factors ( e.g. primer tRNA). It is still not clear though why reverse transcriptase activity in Ty 3 requires processing while processing is irrelevant for the Ty 1 enzyme. The Problem of Transpositional Dormancy: Why Don't Ty Elements Jump?-The longest standing conundrum in Ty 1 phenomenology is the fact that a modest increase in transcription of Ty 1 elements, as when a Ty driven by an active heterologous promoter is introduced into cells, yields a disproportionate increase in transposition (7) . Two models have been proposed to explain this problem (49) . First, defective Ty elements could interfere with transposition either by accumulating in place of active elements or by interfering in trans with the transposition of active elements, a dominant negative effect. Since most Ty 1 and Ty 2 elements are transpositionally competent (49, 50) this cannot be the case. Second, an endogenous transpositional inhibitor might block protein synthesis, or processing, or interfere with Ty-encoded enzymatic activities. Overexpressing a Ty transcript in this model would titrate the inhibitor. The inhibitor cannot be a translational repressor since overexpression causes only the expected proportional increase in translation (51) .

Inefficient Protein Processing Causes Transpositional Dormancy

Sandmeyer (17) has explained the inability of PR mutants to complement differently by considering the need for PR to dimerize to become activated (52) . If PR is only active as a dimer then the PR mutants could have a dominant negative effect (Fig. 3 ). However, dimerization is probably not sufficient to activate PR. Mutations that fuse the TYA and TYB genes into one open reading frame express only the TYA-TYB fusion protein. In both Ty 1(37) and Ty 3(38) the expressed polyprotein remained unprocessed, and neither protein would form VLPs. In both cases, wild-type processing and transposition were restored by expressing the capsid protein (CA). This demonstrates that the fusion protein is not defective but rather that its processing requires CA. Therefore, rather than concluding that PR activity is rate-limiting for formation of transpositionally competent VLPs, Sandmeyer concludes that VLP formation rather may be rate-limiting for activation of PR (17) .

It is not clear how activation might depend on VLP assembly. Three types of formal models might explain this dependence. First, there could be a true allosteric interaction in which association between a TYA-TYB dimer and TYA monomers induces a change in the structure of PR that activates it. Second, PR may actually be activated in the dimer, but its low concentration in the cell might make it unable to process significant amounts of substrate. In a VLP the effective concentration of PR and its substrates would be very high, accelerating processing. Third, VLP formation may occur in competition with some other fate, for example proteolytic degradation. This would both limit the amount of the protein which could accumulate so that even though PR might be activated by dimerization the low concentration of protein would reduce the rate of processing. No data on the stability of the Ty 1 fusion protein when not assembled into VLPs has been published, but in the Ty 3 fusion appears to be unstable (38, 46) .

Altering the Stoichiometry of Ty-encoded Proteins Interferes with Proteolytic Processing

Dinman and Wickner (33) explain the effect of altering stoichiometry as interfering with particle assembly. They believe that a dimer of the Gag-Pol fusion protein nucleates formation of the L-A particle (33) and proposed that increasing the proportion of fusion protein might result in nucleating too many particles, none of which are completely formed, while decreasing the fusion protein may drastically decrease dimerization, interfering with nucleation so that fewer particles form.

Studies with Ty elements suggest that the defect is actually at the processing of Ty-encoded proteins (42) . Normally Ty 1 expresses TYA-TYB (p190) at 3% the level of TYA (p58) (42) . Transposition is blocked when the levels of p190 and p58 are equal. At this ratio p190 is incompletely processed (42) ; about half of the TYA-TYB protein accumulates as p160, created by cleavage at the N terminus of PR (see Fig. 4 ). This phenotype is very different from that caused by expression of p190 in the absence of p58, which eliminates all processing, as described above. Apparently, the abnormal ratio of the primary translation products does not inactivate protease but blocks efficient processing of p190. This result is also consistent with the hypothesis that activation of PR requires an association between p190 and p58 or p54. It may be significant that the cleavage that occurs releases the PR, IN, and RT/RH activities from their association with CA and thus their physical connection to the VLP.

(p58) results in abnormal proteolytic processing. The only processing observed is p58 

p54 and p190 

The sensitive response of the transposition to the p190:p58 ratio suggests that Ty elements might use frameshift efficiency to sense changes in cellular physiology. One would predict that since frameshifting is very sensitive to the availability of the particular slowly decoding aminoacyl-tRNAs, changes in aminoacylation should reduce the likelihood of transposition. Balasundaram et al.(54, 55) have demonstrated that changing the relative intracellular concentrations of the polyamines spermidine and putrescine interferes both with +1 frameshifting on a Ty 1-derived site and with transposition of Ty 1. The mechanism of this interference is unclear, though changing the polyamine pools could have a direct effect on ribosome structure (altering rRNA structure? rearranging ribosomal proteins?) or could act indirectly by interfering with tRNA aminoacylation. It is not clear if the ability of Ty 1 to respond in this fashion to changes in polyamine pools has any evolutionary significance or if it is merely the unavoidable effect of the element depending on an unusual translational event for its propagation.

Summary and Perspectives 

Ty transposition is controlled at several post-transcriptional steps: translational elongation, proteolytic processing, and phosphorylation. Since transposition depends on several other post-transcriptional events (packaging of tRNA primers, creation of a cDNA copy of the Ty mRNA, insertion of the cDNA copy into a new chromosomal location) it is likely that we will find that other post-transcriptional events regulate transposition. The existence of Ty 5 demonstrates that at least one other post-transcriptional process can regulate transposition in yeast. Since Ty 5 encodes all of its products from a single open reading frame, it cannot regulate the critical ratio of structural and enzymatic products by translational frameshifting. In this Ty 5 resembles both the elements Tf 1 of Schizosaccharomyces pombe(56) and copia of Drosophila(57) . It remains to be seen how Ty 5 adjusts the concentration of its gag and pol analogs. copia does so by alternative splicing (58) , while Tf 1 appears to degrade excess enzymatic proteins to adjust the gag: pol ratio.() Use of yeast genetics should allow the identification of host-encoded factors responsible for post-transcriptional control of transposition.

FOOTNOTES

*

This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995.

§

To whom correspondence should be addressed. Tel.: 410-455-3018; Fax: 410-455-3875; Internet: farabaug@umbc.edu.

D. Voytas, personal communication.

The abbreviations used are: LTR, long terminal repeat; VLP, virus-like particle; RT/RH, reverse transcriptase/RNase H; IN, integrase; PR, protease; CA, capsid.

The genes of Ty 1, Ty 2, and Ty 4 are distinguished by a numeral corresponding to the type of element, e.g.TYA1 for the TYA gene from Ty 1. The analogous genes in Ty 3 are termed GAG3 and POL3, which more accurately identifies their relatedness to their retroviral analogs. In addition, Ty 5 includes only one gene that encodes analogs of both retroviral gag and pol. In this review I will refer to the gag analogs collectively as TYA and the pol analogs as TYB; when referring to the Ty 3 genes specifically I will use their correct names.

H. Levin, personal communication.

 

 

ACKNOWLEDGEMENTS

Thanks to Dr. Dan Votyas and Dr. Henry Levin for communicating results before publication and to Dr. Suzanne Sandmeyer for critical reading of the manuscript.

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