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.