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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.

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The influence of the gag-pol junction region in directing pol expression. The Tca2 gag-pol junction region was cloned in between translationally-fused lacZ and luc genes in the dual reporter vector pAC98-U. Panel A: the graph shows the normalised level of downstream luc gene expression measured in cells transformed with the vectors indicated in the 3' deletion series (see panel B). Luciferase expression was first calculated as the ratio of luciferase to β-galactosidase levels, which was then expressed as a percentage of the same ratio measured using the parental vector (pAC98-U; control lacZ-luc). Bars represent means of independent transformants +/- 1 standard deviation (n = 3). Constructs tested all contain 5xEK repeats upstream of the gag stop codon (see Figure 1).
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Figure 2: The influence of the gag-pol junction region in directing pol expression. The Tca2 gag-pol junction region was cloned in between translationally-fused lacZ and luc genes in the dual reporter vector pAC98-U. Panel A: the graph shows the normalised level of downstream luc gene expression measured in cells transformed with the vectors indicated in the 3' deletion series (see panel B). Luciferase expression was first calculated as the ratio of luciferase to β-galactosidase levels, which was then expressed as a percentage of the same ratio measured using the parental vector (pAC98-U; control lacZ-luc). Bars represent means of independent transformants +/- 1 standard deviation (n = 3). Constructs tested all contain 5xEK repeats upstream of the gag stop codon (see Figure 1).

Mentions: In fact, the Tca2 gag-pol junction region did allow expression of the downstream luc reporter, but to a far greater extent than anticipated. Luciferase expression levels measured using the dual reporter vector containing cloned wild-type Tca2 sequences (with 5EK repeats) were 120% of those measured with a control vector in which the TGA gag stop codon had been replaced with a TGT sense codon (pEF7 and pEF8; Figure 2). Similar results were obtained for other cloned gag-pol junctions containing three or four EK repeats (data not shown). In contrast, inserting a control TAA stop codon between the two reporter genes generated the expected 0.3% readthrough, typical of ordinary stop codons with low level 'leakiness' (pUAA; Figure 2).


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 influence of the gag-pol junction region in directing pol expression. The Tca2 gag-pol junction region was cloned in between translationally-fused lacZ and luc genes in the dual reporter vector pAC98-U. Panel A: the graph shows the normalised level of downstream luc gene expression measured in cells transformed with the vectors indicated in the 3' deletion series (see panel B). Luciferase expression was first calculated as the ratio of luciferase to β-galactosidase levels, which was then expressed as a percentage of the same ratio measured using the parental vector (pAC98-U; control lacZ-luc). Bars represent means of independent transformants +/- 1 standard deviation (n = 3). Constructs tested all contain 5xEK repeats upstream of the gag stop codon (see Figure 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The influence of the gag-pol junction region in directing pol expression. The Tca2 gag-pol junction region was cloned in between translationally-fused lacZ and luc genes in the dual reporter vector pAC98-U. Panel A: the graph shows the normalised level of downstream luc gene expression measured in cells transformed with the vectors indicated in the 3' deletion series (see panel B). Luciferase expression was first calculated as the ratio of luciferase to β-galactosidase levels, which was then expressed as a percentage of the same ratio measured using the parental vector (pAC98-U; control lacZ-luc). Bars represent means of independent transformants +/- 1 standard deviation (n = 3). Constructs tested all contain 5xEK repeats upstream of the gag stop codon (see Figure 1).
Mentions: In fact, the Tca2 gag-pol junction region did allow expression of the downstream luc reporter, but to a far greater extent than anticipated. Luciferase expression levels measured using the dual reporter vector containing cloned wild-type Tca2 sequences (with 5EK repeats) were 120% of those measured with a control vector in which the TGA gag stop codon had been replaced with a TGT sense codon (pEF7 and pEF8; Figure 2). Similar results were obtained for other cloned gag-pol junctions containing three or four EK repeats (data not shown). In contrast, inserting a control TAA stop codon between the two reporter genes generated the expected 0.3% readthrough, typical of ordinary stop codons with low level 'leakiness' (pUAA; Figure 2).

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