Limits...
Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.

Forbes EM, Nieduszynska SR, Brunton FK, Gibson J, Glover LA, Stansfield I - BMC Mol. Biol. (2007)

Bottom Line: Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared.This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass.However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Aberdeen, School of Medical Sciences, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. eforbes@email.arizona.edu

ABSTRACT

Background: In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot.

Results: The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA.

Conclusion: This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

Show MeSH

Related in: MedlinePlus

The Tca2 long terminal repeat promoter, but not the gag-pol junction promoter, is heat-shock inducible. Either the Tca2 LTR promoter, or the newly identified gag-pol junction promoter, was cloned upstream of a promoter-less copy of the S. thermophilus lacZ gene integrated into the C. albicans genome at the ADE2 locus. β-galactosidase specific activities were measured in lysates from cultures growing at 30°C (solid bars) or after growth at 23°C followed by a two-hour heat-shock at 37°C (hatched bars). The gag-pol junction promoter activity was assayed in this way using either gag TGA variant (gag-TGA-pol) or TGT sense codon (gag-TGT-pol). The specific activity of the junction promoter constructs is indicated on the bar chart as a percentage of the activity directed by the LTR promoter (100%). Bars represent the means of three independent cultures. Error bars represent +/- 1 standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2194720&req=5

Figure 5: The Tca2 long terminal repeat promoter, but not the gag-pol junction promoter, is heat-shock inducible. Either the Tca2 LTR promoter, or the newly identified gag-pol junction promoter, was cloned upstream of a promoter-less copy of the S. thermophilus lacZ gene integrated into the C. albicans genome at the ADE2 locus. β-galactosidase specific activities were measured in lysates from cultures growing at 30°C (solid bars) or after growth at 23°C followed by a two-hour heat-shock at 37°C (hatched bars). The gag-pol junction promoter activity was assayed in this way using either gag TGA variant (gag-TGA-pol) or TGT sense codon (gag-TGT-pol). The specific activity of the junction promoter constructs is indicated on the bar chart as a percentage of the activity directed by the LTR promoter (100%). Bars represent the means of three independent cultures. Error bars represent +/- 1 standard deviation.

Mentions: C. albicans integrants for each of the constructs were grown in liquid culture, either at a constant temperature of 30°C, or at a temperature of 23°C, followed by a 2-hour heat-shock at 37°C. The latter condition replicates that under which Tca2 is reported to retrotranspose actively [29]. The results show that at a constant 30°C, the junction promoter has an activity 4.9% of that of the LTR promoter (Figure 5). This activity ratio would produce a ratio of gag to gag-pol proteins of 20:1, consistent with molar ratios required in other retroelements to generate active retroviruses, although there is no evidence that Tca2 is active at 30°C. Under heat shock conditions, transcription from the LTR promoter increased five-fold, consistent with the reported increase in retrotransposition under these conditions (Figure 5). However, the junction promoter activity was unaffected by heat-shock, meaning that under conditions where the retrotransposon is reportedly most active, the activity of the junction promoter was only 1.2% of that of the LTR promoter. This departs significantly from gag-pol ratios reportedly required to produce active retrotransposition. These results therefore challenged the hypothesis that retrotransposition is dependent upon the newly identified junction promoter driving expression of a pol-only mRNA template from which Tca2 pol proteins are translated, and instead favour the hypothesis that the LTR promoter somehow directs pol synthesis.


Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2.

Forbes EM, Nieduszynska SR, Brunton FK, Gibson J, Glover LA, Stansfield I - BMC Mol. Biol. (2007)

The Tca2 long terminal repeat promoter, but not the gag-pol junction promoter, is heat-shock inducible. Either the Tca2 LTR promoter, or the newly identified gag-pol junction promoter, was cloned upstream of a promoter-less copy of the S. thermophilus lacZ gene integrated into the C. albicans genome at the ADE2 locus. β-galactosidase specific activities were measured in lysates from cultures growing at 30°C (solid bars) or after growth at 23°C followed by a two-hour heat-shock at 37°C (hatched bars). The gag-pol junction promoter activity was assayed in this way using either gag TGA variant (gag-TGA-pol) or TGT sense codon (gag-TGT-pol). The specific activity of the junction promoter constructs is indicated on the bar chart as a percentage of the activity directed by the LTR promoter (100%). Bars represent the means of three independent cultures. Error bars represent +/- 1 standard deviation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2194720&req=5

Figure 5: The Tca2 long terminal repeat promoter, but not the gag-pol junction promoter, is heat-shock inducible. Either the Tca2 LTR promoter, or the newly identified gag-pol junction promoter, was cloned upstream of a promoter-less copy of the S. thermophilus lacZ gene integrated into the C. albicans genome at the ADE2 locus. β-galactosidase specific activities were measured in lysates from cultures growing at 30°C (solid bars) or after growth at 23°C followed by a two-hour heat-shock at 37°C (hatched bars). The gag-pol junction promoter activity was assayed in this way using either gag TGA variant (gag-TGA-pol) or TGT sense codon (gag-TGT-pol). The specific activity of the junction promoter constructs is indicated on the bar chart as a percentage of the activity directed by the LTR promoter (100%). Bars represent the means of three independent cultures. Error bars represent +/- 1 standard deviation.
Mentions: C. albicans integrants for each of the constructs were grown in liquid culture, either at a constant temperature of 30°C, or at a temperature of 23°C, followed by a 2-hour heat-shock at 37°C. The latter condition replicates that under which Tca2 is reported to retrotranspose actively [29]. The results show that at a constant 30°C, the junction promoter has an activity 4.9% of that of the LTR promoter (Figure 5). This activity ratio would produce a ratio of gag to gag-pol proteins of 20:1, consistent with molar ratios required in other retroelements to generate active retroviruses, although there is no evidence that Tca2 is active at 30°C. Under heat shock conditions, transcription from the LTR promoter increased five-fold, consistent with the reported increase in retrotransposition under these conditions (Figure 5). However, the junction promoter activity was unaffected by heat-shock, meaning that under conditions where the retrotransposon is reportedly most active, the activity of the junction promoter was only 1.2% of that of the LTR promoter. This departs significantly from gag-pol ratios reportedly required to produce active retrotransposition. These results therefore challenged the hypothesis that retrotransposition is dependent upon the newly identified junction promoter driving expression of a pol-only mRNA template from which Tca2 pol proteins are translated, and instead favour the hypothesis that the LTR promoter somehow directs pol synthesis.

Bottom Line: Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared.This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass.However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Aberdeen, School of Medical Sciences, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. eforbes@email.arizona.edu

ABSTRACT

Background: In the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot.

Results: The hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA.

Conclusion: This is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

Show MeSH
Related in: MedlinePlus