In this paper we demonstrate that a major block to transposition at high temperature is the inhibition of PR activity, exacerbated by a decrease in overall Ty1 protein levels. The decrease in overall Gag-Pol protein level observed at 37°C could be a consequence of the decreased PR activity or it could be due to some unrelated change in cellular physiology. When expressed in E. coli
, recombinant Ty1 PR is nonfunctional at high temperature, suggesting an intrinsic temperature sensitivity. In yeast, cleavage of the Gag-Pol-p199 polyprotein is completely blocked at high temperature, and cleavage of the Gag protein is slightly reduced in some strains. In VLPs formed at high temperature, exogenous RT activity is significantly reduced but detectable, presumably reflecting a decrease in total RT protein, as this exogenous RT activity is not inherently temperature sensitive in vitro. Endogenous RT activity, determined by measuring Ty1 cDNA production, decreases with increasing growth temperature, consistent with reduced RT amounts and activity, and is undetectable at 37°C. Thus, the exogenous RT activity detected in purified VLPs is insufficient to produce detectable cDNA in vivo. This conclusion agrees with earlier studies in which PR-deficient mutants failed to synthesize cDNA (21
). We hypothesize that a temperature-induced conformational change in the Ty1 Gag-Pol polyprotein reduces the activity of both PR and RT, resulting in nearly undetectable levels of transposition. It is likely that the reduction in Ty1 protein levels at 37°C also contributes to the severe transposition defect, as Ty1 transposition does not necessarily correlate with the amount of detected protein. Curcio and Garfinkel previously showed that expression of a galactose-inducible Ty1 element increases transposition efficiency at the posttranslational level, and their studies suggested that the activity of endogenous Ty1 elements may be limited by the levels of PR activity (9
). Correspondingly, we have shown that a major defect in high-temperature transposition is posttranslational. It is possible that a limiting factor for transposition falls below a threshold level at temperatures above 32°C, contributing to the rapid drop-off in transposition.
A recently published study determined the necessity of each of the Ty1 cleavage sites for Ty1 transposition (21
). Proteolytic processing of the three Ty1 cleavage sites occurs in a regulated and semiordered manner. Gag/PR must be cleaved first, after which cleavage of the other two sites occurs without required order. Blockage of either of the cleavage sites in Pol (PR/IN and IN/RT), independently or in combination, did not affect cDNA production in these mutants, and the affected site(s) shows a cleavage defect. However, blocking the Gag*PR cleavage site abolished cleavage at all three sites and also blocked cDNA production. A PR−
active-site mutant, in which no processing occurs, was also defective in cDNA synthesis. We found that, in VLPs formed at high temperature, a different phenotype was observed. Processing at the two Pol sites (PR/IN and IN/RT) was completely abolished. However, unlike what was found for PR−
and Gag*PR processing site mutants, processing at high temperature in yeast was not completely blocked, as indicated by the presence of processed Gag-p45 protein from the Gag-p49 primary translation product. This processing of p49 required active Ty1 PR. Taken together, these results point to a context-dependent decrease in PR activity wherein Gag-Pol is not processed at the Gag-PR junction but Gag-p49 is processed. Curiously, both of these processing events occur at the same scissile bond located between histidine 401 and asparagine 402 (Fig. ).
Since we observe processing of Gag, it appears that VLPs formed at high temperature harbor an additional defect, other than processing, that blocks cDNA production. Production of cDNA is gradually reduced as temperature increases. A mild increase in temperature (to 30°C) reduces the amount of cDNA produced, although Gag-Pol-p199 processing occurs readily at this temperature. Thus, as temperature increases, VLPs become less efficient at producing cDNA. The observed reduction of RT activity was still expected to produce a small but detectable amount of residual cDNA synthesis at 37°C. However, we failed to detect any cDNA. The cDNA synthesis defect might therefore be exacerbated by the slight decrease in Gag processing. In summary, it is likely that the temperature sensitivity of Ty1 retrotransposition reflects the combined effects of deficiencies in both proteolytic processing and in vivo cDNA synthesis.
Previous studies have shown that PR−
mutants are defective in cDNA production, not because RT is inactive but because of a defect in accessing the endogenous primer or template or a defect in forming dimeric RNA or both (13
). The initial step to cDNA synthesis in Ty1 is the annealing of the host-encoded tRNAiMet
primer to the primer-binding site (PBS) on the Ty RNA. Data from studies of PBS region mutants suggest that formation of the primer/template complex is a temperature-sensitive step in transposition (18
). A G:U mismatch introduced into the primer/template complex reduces transposition activity to approximately one-third wild-type levels at 27°C. Conversely, extension of the PBS complementarity region from 10 to 12 nt conferred a slight temperature resistance to transposition (17
). At 34°C, transposition levels of an element containing a 12-nt PBS are nearly 100-fold greater than that of a wild-type element. However, transposition in these mutants is still substantially less than that seen at 22°C. Therefore, the lack of cDNA at high temperature may be attributed only in part to reduced efficiency of primer/template formation. It is also possible that temperature-induced conformational changes in the template/primer complexes that form at high temperature further reduce the efficiency of RT initiation.
Transposition is very sensitive to the conformation of the primer/template complex. Introduction of a G·U mismatch, while not expected to significantly affect primer/template annealing, does result in a slightly temperature-sensitive phenotype. Additionally, a single mismatch in the primer/template complex reduces transposition dramatically (16
). Thus, temperature may have an effect on priming at two steps: high temperature may reduce the efficiency of primer/template annealing and may affect the conformational structure of complex that forms. Recent studies suggest that the amino terminus of the Ty1 PR plays an important role in the initiation of reverse transcription in vivo and that Gag is involved in primer-template annealing (8
Primer/template formation, the initial step in cDNA formation, is followed by RT-mediated DNA synthesis. The formation of cDNA in VLPs is a measure of the endogenous activity of RT. In this study, we also measured the ability of RT to synthesize DNA from an exogenous primer/template complex. Formation of this exogenous oligo(dG)/poly(C) primer/template complex is not temperature dependent. Our data show that RT activity is not inherently temperature sensitive. RT activities at 22 and 37°C in VLPs formed at 22°C are comparable. However, RT activity is greatly reduced in VLPs formed at high temperature, regardless of whether the in vitro assay is performed at 22 or 37°C. PR−
mutant VLPs prepared at 22°C have readily detectable RT activity in these assays, indicating that processing is not required for activity and that RT is functionally active as part of the Gag-Pol-p199 polyprotein (21
). These results argue that high temperature affects the conformation of the RT protein attained during synthesis, rendering it inactive. That the RT activity is not inherently temperature sensitive suggests that RT folding occurs during particle formation as part of the polyprotein and that RT, once folded, remains stable. Thus, particle formation at high temperature could result in an inactive conformation. Folding is likely to be a complex process, affected by interactions with other Ty1 or host factors (29
). An alternative hypothesis is that exogenous RT activity is reduced simply because there is markedly less RT protein in VLPs formed at high temperature. We normalized to Gag proteins in the exogenous RT assays, and activity was still reduced 15-fold. Therefore, the inactivation of RT is significant.
The Gag-Pol-p199 protein is readily detectable at 37°C, but the overall levels of Pol protein products are reduced relative to levels seen at 22°C. Our immunoblot analyses suggest that the reduction of total Pol protein products at 37°C is greater than the reduction in Gag protein levels. We tested the effect of temperature on frameshifting and found that frameshifting is modestly affected, being approximately twofold more efficient at 37°C than at 22°C. A more efficient frameshifting mechanism would be expected to yield relatively more Pol protein product, not less. However, our findings resemble the results of a previous study on the effects of increased frameshifting efficiency. Frameshifting is mediated by a ribosomal stalling event caused by a rare tRNAArg
codon at the +1 frameshifting site. Deletion of the gene for this tRNA increases frameshifting 3- to 17-fold (15
). Interestingly, this mutation did not result in an accumulation of Pol products; rather the particles displayed a processing defect in that processed IN is undetectable in the mutant strain. As for VLPs made at high temperature, the processing of Gag in this mutant was not affected. Thus, an increase in frameshifting results in a processing defect, perhaps due to reduced PR activity in the context of particles forming in the presence of skewed Gag/Gag-Pol protein ratios. It therefore remains possible that a slight increase in frameshifting efficiency at high temperature could contribute to the observed Pol processing defect.
We have shown that the temperature sensitivity of transposition is due to a reduction in both PR and RT activity, resulting in a profound processing defect and a lack of any detectable cDNA in VLPs formed at 37°C. We hypothesize that aberrant folding at high temperature adversely affects the activities of both of these enzymes. Additionally, reduced protein levels, primer/template formation, and frameshifting effects may contribute to the phenotype of high-temperature VLPs. It is unknown whether the activity of IN is also affected by high temperature in vivo. Although reduced PR cleavage and RT function are major blocks to transposition at high temperature, the complete inhibition may well be due to the additive effects of defects in other steps as well. Such a complex control system allows adjustment to multiple environmental signals. Host mutants that partially restore transposition and processing at high temperature have been isolated (J. B. Keeney, unpublished data). Further characterization of these mutants will identify additional host cell-mediated events in the complex control of transposition.