Ty1 is one of five endogenous retrotransposons of the yeast Saccharomyces cerevisiae
. The life cycle of Ty1 begins with transcription of the genomic element and translation of the resulting mRNA. Like retroviruses, Ty1 encodes a protease (PR) that processes the element-encoded proteins (1
). Ty1 Gag and Gag-Pol proteins, together with Ty1 mRNA, form virus-like particles (VLPs), which are essential transposition intermediates (8
). Reverse transcription of Ty1 mRNA takes place in VLPs. The newly synthesized cDNA enters the nucleus and then the yeast genome via an integration reaction (3
Viruses and the closely related retrotransposons are under selective pressure to maintain a small genome. A number of unique strategies have evolved to ensure that all of the functions necessary for completion of their life cycle are carried out. Host protein functionalities may be usurped, obviating the need for a similar, element-encoded protein.
A genome streamlining strategy used by most long terminal repeat-containing retroelements is programmed ribosomal frameshifting. An mRNA that contains a frameshifting signal can encode multiple proteins, typically Gag and Gag-Pol, in a predetermined stoichiometric ratio that is determined by the efficiency of the frameshift signal. A frameshift-containing retroelement does not require separate promoters to drive the transcription of two messages and thereby increases the information content of its genome with minimal expense. In addition, the common sequences present in Gag and Gag-Pol direct the assembly of the Pol proteins into the virion or VLP.
A 7-nucleotide signal in Ty1 mRNA is necessary and sufficient for directing ribosomal frameshifting (2
) and can function translationally in various heterologous contexts. This frameshift signal is required for retrotransposition and is thought to secure the appropriate Gag and Gag-Pol stoichiometry. The frameshifting signal is approximately 5 to 10% efficient and therefore results in a 10- to 20-fold excess of the structural protein Gag with respect to Gag-Pol (6
). The Gag-Pol polyprotein contains the enzymatic components required for Ty1 replication.
Ty1 mRNA can have two translational fates. In the more common scenario, in which the frameshift signal is read through by the translating ribosome, a 49-kDa Gag species is made and processed in trans
to a 45-kDa Gag species with concomitant liberation of its carboxy-terminal 40 amino acids. The 49-kDa precursor and 45-kDa (CA) processed forms migrate with apparent molecular masses of 58 and 54 kDa, respectively. For a complete description of our Ty1 protein nomenclature, see the study by Merkulov et al. (14
). The liberated 40-mer, called the p4 peptide, was suspected to serve some role in particle formation and/or maturation, because mutations in this region result in smaller-than-normal VLPs and severe transposition defects (15
). When frameshifting occurs, a Gag-Pol species is synthesized. Proteolytic processing of Gag-Pol liberates Ty1 PR, integrase (IN), and reverse transcriptase (RT) from the precursor polyprotein (10
). A 45-kDa Gag species, CA, identical to that made from Gag is also produced, and we show here that it is through this processing event that the amino terminus of the PR is defined (Fig. ). Most of the p4 peptide sequence is therefore common to the amino terminus of the PR and the carboxy terminus of 49-kDa Gag.
FIG. 1 Schematic of the p4 region. The Gag and Gag-Pol polyproteins as well as the relevant amino acids are shown. The amino acids encoded fully or in part by the frameshift are underlined. The PR active site residues are in boldface. The PR cleavage sites in (more ...)
Certain mutations in the p4 region common to Gag, p4, and PR block transposition. However, interpretation of this mutant phenotype is complicated by the fact that these mutations simultaneously alter Gag, PR, and p4 sequences. The transposition defect observed in these mutants could be secondary to a loss or gain of function in any or all of these proteins. The functional independence of the frameshift signal suggests a unique manner by which the critical Ty1 protein(s) affected by these mutations can be identified.
In this study, we describe the creation of frameshift transplant mutants of Ty1. These mutants enable us to specifically and unambiguously attribute the deleterious effects of mutations in the p4 region to the Ty1 PR. The p4 peptide, previously thought to be required for transposition, is shown here to be dispensable. We also physically define the amino terminus of the PR by microsequencing and thereby confirm the location of the Gag-PR cleavage site. We confirm that the mutations in this region affect the N terminus of the Ty1 PR; frameshift transplant mutants combined in cis with P4 region mutations are as defective as the same mutants in an otherwise wild-type Ty1 element. Frameshift transplantation is a novel approach that permits the isolated study of one or more proteins encoded by a single mRNA. This approach should prove useful in the study of viruses that are under selective pressure to maintain small, information-dense genomes.