4. Yeast Ty elements resemble retroviruses

16.4 Yeast Ty elements resemble retroviruses

Ty elements comprise a family of dispersed repetitive DNA sequences that are found at different sites in different strains of yeast. Ty is an abbreviation for "transposon yeast." A transposition event creates a characteristic footprint: 5 bp of target DNA are repeated on either side of the inserted Ty element. The frequency of Ty transposition is lower than that of bacterial transposons, ~10 ^V7 V10 ^V8.

Figure 16.12 Ty elements terminate in short direct repeats and are transcribed into two overlapping RNAs. They have two reading frames, with sequences related to the retroviral gag and pol genes.

There is considerable divergence between individual Ty elements. Most elements fall into one of two major classes, called Ty1 and Ty917. They have the same general organization illustrated in Figure 16.12. Each element is 6.3 kb long; the last 330 bp at each end constitute direct repeats, called δ . Individual Ty elements of each type have many changes from the prototype of their class, including base pair substitutions, insertions, and deletions. There are ~30 copies of the Ty1 type and ~6 of the Ty917 type in a typical yeast genome. In addition, there are ~100 independent delta elements, called solo δs.

The delta sequences also show considerable heterogeneity, although the two repeats of an individual Ty element are likely to be identical or at least very closely related. The delta sequences associated with Ty elements show greater conservation of sequence than the solo delta elements, which suggests that recognition of the repeats is involved in transposition.

The Ty element is transcribed into two poly(A)⁺ RNA species, which constitute >5% of the total mRNA of a haploid yeast cell. Both initiate within a promoter in the δ element at the left end. One terminates after 5 kb; the other terminates after 5.7 kb, within the delta sequence at the right end.

The sequence of the Ty element has two open reading frames, expressed in the same direction, but read in different phases and overlapping by 13 amino acids. The sequence of TyA suggests that it codes for a DNA Vbinding protein. The sequence of TyB contains regions that have homologies with reverse transcriptase, protease, and integrase sequences of retroviruses.

The organization and functions of TyA and TyB are analogous to the behavior of the retroviral gag and pol functions. The reading frames TyA and TyB are expressed in two forms. The TyA protein represents the TyA reading frame, and terminates at its end. The TyB reading frame, however, is expressed only as part of a joint protein, in which the TyA region is fused to the TyB region by a specific frameshift event that allows the termination codon to be bypassed (analogous to gag Vpol translation in retroviruses).

Recombination between Ty elements seems to occur in bursts; when one event is detected, there is an increased probability of finding others. Gene conversion occurs between Ty elements at different locations, with the result that one element is "replaced" by the sequence of the other.

Ty elements can excise by homologous recombination between the directly repeated delta sequences. The large number of solo delta elements may be footprints of such events. An excision of this nature may be associated with reversion of a mutation caused by the insertion of Ty; the level of reversion may depend on the exact delta sequences left behind.

A paradox is that both delta elements have the same sequence, yet a promoter is active in the delta at one end and a terminator is active in the delta at the other end. (A similar feature is found in other transposable elements, including the retroviruses.)

Figure 16.13 A unique Ty element, engineered to contain an intron, transposes to give copies that lack the intron. The copies possess identical terminal repeats, generated from one of the termini of the original Ty element.

Ty elements are classic retroposons, transposing through an RNA intermediate. An ingenious protocol used to detect this event is illustrated in Figure 16.13. An intron was inserted into an element to generate a unique Ty sequence. This sequence was placed under the control of a GAL promoter on a plasmid and introduced into yeast cells. Transposition results in the appearance of multiple copies of the transposon in the yeast genome; but they all lack the intron (Boeke et al., 1985).

We know of only one way to remove introns: RNA splicing. This suggests that transposition occurs by the same mechanism as with retroviruses. The Ty element is transcribed into an RNA that is recognized by the splicing apparatus. The spliced RNA is recognized by a reverse transcriptase and regenerates a duplex DNA copy.

Figure 16.3 The genes of the retrovirus are expressed as polyproteins that are processed into individual products.

Figure 16.4 Retroviruses (HIV) bud from the plasma membrane of an infected cell. Photograph kindly provided by Matthew Gonda.
Multiple figure

Figure 16.5 Retroviral RNA ends in direct repeats (R), the free linear DNA ends in LTRs, and the provirus ends in LTRs that are shortened by two bases each.

Figure 16.6 Minus strand DNA is generated by switching templates during reverse transcription.

The analogy with retroviruses extends further. The original Ty element has a difference in sequence between its two delta elements. But the transposed elements possess identical delta sequences, derived from the 5′ delta of the original element. If we consider the delta sequence to be exactly like an LTR, consisting of the regions U3 VR VU5, the Ty RNA extends from R region to R region. Just as shown for retroviruses in Figure 16.3, Figure 16.4, Figure 16.5, Figure 16.6, the complete LTR is regenerated by adding a U5 to the 3′ end and a U3 to the 5′ end.

Transposition is controlled by genes within the Ty element. The GAL promoter used to control transcription of the marked Ty element is inducible: it is turned on by the addition of galactose. Induction of the promoter has two effects. It is necessary to activate transposition of the marked element. And its activation also increases the frequency of transposition of Ty elements on the yeast chromosome. This implies that the products of the Ty element can act in trans on other elements (actually on their RNAs).

Figure 16.14 Ty elements generate virus-like particles. Photograph kindly provided by Alan Kingsman.

Although the Ty element does not give rise to infectious particles, virus Vlike particles (VLPs) accumulate within the cells in which transposition has been induced. The particles can be seen in Figure 16.14. They contain full-length RNA, double-stranded DNA, reverse transcriptase activity, and a TyB product with integrase activity. The TyA product is cleaved like a gag precursor to produce the mature core proteins of the VLP. This takes the analogy between the Ty transposon and the retrovirus even further. The Ty element behaves in short like a retrovirus that has lost its env gene and therefore cannot properly package its genome.

Only some of the Ty elements in any yeast genome are active: most have lost the ability to transpose (and are analogous to inert endogenous proviruses). Since these "dead" elements retain the δ repeats, however, they provide targets for transposition in response to the proteins synthesized by an active element.

Research

Boeke, J. D. et al. (1985). Ty elements transpose through an RNA intermediate. Cell 40, 491-500.