Efficient translational frameshifting occurs within a conserved sequence of the overlap between the two genes of a yeast Ty1 transposon.

Abstract

The Ty (transposon yeast) family of retroviral-like transposons include two genes, TYA and TYB, analogous to the gag andpol genes ofmetazoan retroviruses. TYB lies downstream of TYA, the two genes overlapping by 38 base pairs. The primary translation product of TYB is a TYA/TYB fusion protein whose expression has been inferred to occur by translational frameshifting within the overlap region. We show that the event leading to expression of TYB is very efficient, resulting in 20% read-through into TYB from TYA. We demonstrate that the Ty mRNA is colinear with the DNA sequence of the element, eliminating any pretranslational model for TYB expression. Frameshifting requires no particular sequence of the upstream TYA gene, nor any global RNA structure. Surprisingly, it can be promoted by a 14-base-pair oligonucleotide of the overlap region. The ability of this oligonucleotide to function is inhibited when it is positioned immediately downstream of an initiator AUG. We conclude that the TYB gene is expressed by an efficient ribosomal frameshifting event requiring a small oligonucleotide sequence derived from the TYAITYB overlap region.The Ty (transposon yeast) elements of the yeast Saccharomyces cerevisiae are a family of about 30 dispersed retroviral-like transposons (1, 2). Ty elements include two open reading frames, termed TYA and TYB (3), which are analogous respectively to the gag and pol genes of metazoan retroviruses. The similarity in structure and organization reflects the fact that Ty elements, like retroviruses, replicate via an RNA intermediate during transposition (2). The primary translation product of retroviral pol genes and TYB are fusions to the product ofthe upstream gene, gag and TYA. In the case of most of the avian and mammalian retroviruses the gag, pol, and sometimes pro (encoding retroviral protease) genes overlap; similarly TYB overlaps the last 38-44 base pairs (bp) of TYA. For Rous sarcoma virus (RSV) (4) and mouse mammary tumor virus (MMTV) (5, 6), translational frameshifting has been shown explicitly to produce the fusion peptides. It is likely, given their similarity to retroviruses, that Ty elements also express theirpol analog by translational frameshifts; however, the evidence for this is circumstantial. Nuclease S1 protection experiments eliminate the possibility that TYB expression involves RNA splicing (3,7,8). The experiments are sufficiently sensitive to detect a very small discontinuity between the RNA and the DNA [5-11 nucleotides (nt)]; these experiments do not exclude the possibility that expression of TYB occurs by RNA editing.Wilson et al. (8) have demonstrated that a 125-bp region of the element Tyl-15, extending from a position 7 bp into the overlap through a Bgl II site 94 bp downstream ofthe overlap, is sufficient to promote normal TYAITYB expression. They also showed that a deletion removing the first 24 nt of the overlap eliminates TYAITYB expression. They speculated that a short region of the overlap that is conserved between the TyJ and Ty2 class elements,...

Ribosomal frameshifting in plants: a novel signal directs the −1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

“…For example, in animal retroviruses, expression of the viral RNA-dependent DNA polymerase (reverse transcriptase) occurs by -1 ribosomal frameshifting (Jacks and Varmus, 1985;Jacks et al, 1987Jacks et al, , 1988aMoore et al, 1987;Wilson et al, 1988). Frame-shifting apparently also operates in the expression of reverse transcriptase for several retrotransposons [Tyl (Mellor et al, 1985;Wilson et al, 1986;Clare et al, 1988), Ty912 (Clare and Farabaugh, 1985), 17.6 (Saigo et al, 1984) and gypsy (Marlor et al, 1986)]. Other viruses may also depend on frameshifting for the expression of some of their genes as has been documented for infectious bronchitis coronavirus Oxford University Press (IBV) in the translation of the Fl and F2 proteins (Brierly et al, 1987(Brierly et al, , 1989.…”

Escherichia coli RNase HI inhibits murine leukaemia virus reverse transcription in vitro and yeast retrotransposon Ty1 transposition in vivo

“…5), in which a capsid-E. coli RNase HI fusion protein was produced in higher levels as a consequence of fusing the rnhA gene to the capsid protein at a point prior to the frame-shift position (Clare & Farabaugh 1985) and, thereby, increasing the probability that the capsid-E. coli RNase HI fusion protein would be incorporated into VLPs during assembly. All the capsid molecules generated from the direct fusion plasmid would be expected to include E. coli RNase HI, increasing the amount of the capsid-E. coli RNase HI fusion protein fivefold over the frame-shift version (Clare et al 1988). When transposition was measured using the direct fusion proteins, nearly 100% inhibition was observed for both wild-type [pWPM209.8(WT-DF)] and P144 L (rnhA-1 ) [pWPM213(P144L-DF)] proteins.…”

“…5. The ratio of capsid to polymerase is regulated, in part, by differential expression of the capsid and capsid-polymerase fusion protein via a frame-shift in translation between the capsid and polymerase region Clare et al 1988). Therefore, two different types of constructs were examined (Fig.…”

Glucose Signaling Pathway and Growth Conditions Regulate Gene Expression in Retrotransposon Ty2

“…It was used as a control fusion gene in this research. Ty2 frameshift and Ty2 frame fusion reporter plasmids are 2-µM-URA3-based shuttle vectors (Clare et al, 1988;Belcourt and Farabaugh, 1990). In Ty2 frameshift plasmids, the Ty2 frameshift site is fused to the E. coli lacZ gene in the + 1 reading frame.…”

“…Ty2 frame fusion construct does not contain the frameshift site. In this expression vector, translation of the TYAlacZ fusion protein does not require a frameshift and takes place at zero frames (Clare et al, 1988;Belcourt and Farabaugh, 1990). Plasmids were transformed into the competent yeast cells as described previously using the lithium acetate method (Ito et al, 1983).…”