mRNA secondary structure melting during translation elongation in yeast
Two assay systems were designed and constructed in this study in order to investigate mRNA secondary structure melting during translation elongation in S. cerevisiae. The first, where stable secondary structures were placed within a lacZ reporter mRNA, was used to demonstrate that such structures are resolved efficiently, as the insertion of a stable stem-loop of -66 kcal/mol into the lacZ coding region only reduced translation efficiency by 40%. It was further demonstrated that the effect of secondary structures in open reading frames are not additive, as two stem-loops of -45 kcal/mol adjacent to one another did not decrease translation efficiency, to the extent expected of a single stem-loop of -90 kcal/mol. The L-A d.s. RNA virus RNA pseudoknot had no effect on translation elongation. A second assay, based on -1 frameshifting upstream of RNA secondary structure elements was used to test candidate genes for a role in mRNA secondary structure melting during translation elongation. It was demonstrated that two cytoplasmic RNA helicases, Ded1p and Dbp5p are not involved in this process. In contrast to previous work (Cui, et. al., 1996; EMBO 15:5726-5736) this study revealed that Upf1p, an RNA helicase involved in nonsense-mediated decay, played no role in -1 ribosomal frameshifting. Environmental effects on mRNA secondary structure melting were investigated by measuring secondary structure melting at 18oC, using the -1 frameshift assay. Contrary to expectations, -1 frameshifting was not enhanced by low temperature, perhaps because a helicase-like activity is induced at 18oC. Proteomic analysis revealed that increased levels of Gdh1p are associated with polyribosomes at 18oC compared to 37oC. Gdh1p interacts with Lsm1p, a component of an mRNA decapping complex, in a two-hybrid screen.