Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene

Abstract

The 5-region of the TIF4631 gene of Saccharomyces cerevisiae (encoding the translation initiation factor eIF4G1) was reported earlier to harbor a very active internal ribosome entry site (IRES) allowing for internal initiation of translation of TIF4631 mRNA. Here, we report the presence of a promoter in the region −112 to −36 relative to the translation initiation codon of the TIF4631 gene. This promoter stimulates transcription from a start site at position −36 and generates an mRNA that is actively translated in vitro and able to sustain growth of yeast cells in vivo as the only source of eIF4G. The data show that the IRES activity reported earlier is due to this promoter. On the contrary, the presumed IRES represents a strongly inhibitory element for translation in vitro.

Reevaluation of the conclusion that IRES-activity reported within the 5′ leader of the TIF4631 gene is due to promoter activity

“…Keywords: IRES; TIF4631; eIF4G; translation; mRNA Verge et al (2004), in the February issue of RNA, reported that contrary to our earlier publication (Zhou et al 2001), the 5Ј leader of the TIF4631 mRNA does not enhance translation initiation or contain an IRES. The authors assert that they have identified a promoter, located 36-112 nucleotides upstream of the translation initiation site, and concluded in their abstract, "The data show that the IRES activity reported earlier is due to this promoter".…”

“…However, Verge and colleagues recently suggested that this sequence does not facilitate translation initiation, but inhibits translation in vitro and has promoter activity when tested in cells. We disagree with these conclusions and respond by showing that the data are most consistent with an internal initiation mechanism.Keywords: IRES; TIF4631; eIF4G; translation; mRNA Verge et al (2004), in the February issue of RNA, reported that contrary to our earlier publication (Zhou et al 2001), the 5Ј leader of the TIF4631 mRNA does not enhance translation initiation or contain an IRES. The authors assert that they have identified a promoter, located 36-112 nucleotides upstream of the translation initiation site, and concluded in their abstract, "The data show that the IRES activity reported earlier is due to this promoter".…”

“…The fact that the translation of the second cistron did not increase when the transcription of the dicistronic mRNA was induced (Verge et al 2004, Fig. 2) appears at first to be definitive evidence that the TIF4631 5Ј leader does not function as an IRES.…”

“…Inasmuch as the dicistronic mRNA was easily detected in our study, a shorter monocistronic transcript should also have been easily identified; however, we found no evidence for such a transcript, even when our Northern blots were overexposed. Verge et al (2004) reported promoter activity within the 5Ј leader of the TIF4631 mRNA; however, they did not directly test for this activity, for example, by testing the fragment in question in a promoterless vector or with Northern blots. Instead, indirect methods were used to assess promoter activity.…”

“…They also identified two longer primer extension products (−390 and −580), one of which corresponds to that previously reported by Goyer et al (1993), and that was used in our study. In their discussion, Verge et al (2004) concede that "we cannot exclude that additional longer forms of the TIF4631 mRNA are produced and perhaps translated by internal initiation". However, in their abstract, they contradicted this by strongly ruling out these possibilities.…”