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

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.

Figure 1

The Tca2 genome structure. The Tca2 retrotransposon comprises gag and pol genes. Within pol are arranged the protease, integrase and reverse transcriptase/RNAse H activities (PRO, IN and RT respectively). Gag and pol ORFs, both in the zero translation frame, are separated by a TGA stop codon. The positions of the potential pseudoknot and polymorphic repeat are shown (see text for details).

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).

Figure 3

Expression of pol directed by the isolated gag-pol junction region is predominantly independent of gag translation. The Tca2 gag-pol junction region was cloned in between translationally-fused lacZ and luc genes in the dual reporter vector pAC98 (Materials and Methods). Panel A: the bar chart shows the normalised level of downstream luc gene expression in constructs transformed with the parental vector pAC98 (control lacZ-luc), wild-type Tca2 stop and sense variants (pGRE1 and 2), junction variants with three in-frame stop codons or three CAA (Gln) codons, respectively, cloned at the 3' end of lacZ (pGRE5 and 6), junction variants with the luc ORF placed in the -1 frame with respect to lacZ (pGRE 7 and 8), and junction variants with three -1 frame AUG codons followed immediately by a -1 frame stop codon, introduced at the 5' end of the luc ORF (pGRE 3 and 4). Constructs are depicted schematically in panel B, and panel C indicates the positions of relevant natural AUG codons found in the 0 (*) and +1 frames (+) in the wildtype, non-mutagenised, Tca2 junction region (see text for details). Bars represent means normalised luciferase activities of independent transformants +/- 1 standard deviation (n = 3).

Figure 4

The Tca2 gag-pol junction region contains a novel promoter activity. The SV40 promoter that directs expression of the lacZ-luc translational fusion in the pAC98 vector was deleted in a series of Tca2 gag-pol junction constructs to determine if the Tca2 gag-pol junction region contained a promoter activity. Panel A portrays the specific luciferase expression level in cells transformed with (i) the control parental vector pFB3 lacking an SV40 promoter and any Tca2 sequence (ii) SV40 promoter deletion constructs containing stop (TGA) and sense (TGT) variants of the Tca2 junction region (pFB1 and pFB2 respectively). The SV40 promoter that directs expression of the lacZ-luc translational fusion in the pAC98 vector was replaced with the strong S. cerevisiae TEF1 promoter to determine the extent of any gag stop codon readthrough. Panel B: the bar chart shows the normalised level of downstream luc gene expression in cells transformed with (i) the parental vector pAC98-TEF (control lacZ-luc) (ii) a construct containing a TAA stop codon in a good termination context at the 3' end of the lacZ ORF (pUAA-TEF) (iii) constructs containing stop (TGA; pJB1-TEF) and sense (TGT; pJB2-TEF) variants of the full-length Tca2 junction region (nt. 826–1131; Figure 1) (iv) a construct containing stop (TGA; pGRE1-TEF) and sense (TGT; pGRE2-TEF) variants of the Tca2 junction region with a partial 3' deletion. Bars represent means of independent transformants +/- 1 standard deviation (n = 3). Constructs are depicted schematically in panel C.

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.

Figure 6

Tca2 pol protein expression is directed by LTR promoter activity in C. albicans. The pol ORF of a genomic copy of C. albicans Tca2 was tagged at its 3' end immediately before the pol stop codon with Protein A coding sequence. Panel A; proteins from the untransformed host strain (CAI-4) and a pol-Protein A integrant (ProA-Int) were separated (SDS-PAGE), western-blotted, and blots probed with anti-Protein A antibody. Arrowhead indicates the migration position of a 76 kDa protein induced under heat shock conditions, present in the integrant but not the parental strain CAI-4. Results are typical of at least three experiments involving independent Protein A integrant isolates. Panel B; a replicate gel was Coomassie Blue-stained to show equivalent lane loadings. Panel C; the anti-Protein A antibody specificity was confirmed by probing a control blot of untransformed CAI-4 lysate and that from an S. cerevisiae strain expressing TAP-tagged eIF4A translation factor [59]. The 65 kDa protein detected by the antibody (indicated by arrowhead) represents eIF4A (44.6 kDa) fused to the TAP tag (20 kDa), the latter containing Protein A.