SITE-DIRECTED MUTATIONS REVEAL LONG-RANGE COMPENSATORY INTERACTIONS IN THE ADH GENE OF DROSOPHILA-MELANOGASTER

Long-range interactions between the 5' and 3' ends of mRNA molecules h ave been suggested to play a role in the initiation of translation and the regulation of gene expression. To identify such interactions and to study their molecular evolution, we used phylogenetic analysis to g enerate a model of mRNA higher-order structure in the Adh transcript o f Drosophila melanogaster. This model predicts long-range, tertiary co ntacts between a region of the protein-encoding sequence just downstre am of the start codon and a conserved sequence in the 3' untranslated region (UTR). To further examine the proposed structure, site-directed mutations were generated in vitro in a cloned D. melanogaster Adh gen e, and the mutant constructs were introduced into the Drosophila germ line through P-element mediated transformation. Transformants were spe ctrophotometrically assayed for alcohol dehydrogenase activity. Our re sults indicate that transformants containing a silent mutation near th e start of the protein-encoding sequence show an approximate to 15% re duction in alcohol dehydrogenase activity relative to wild-type transf ormants. This activity can be restored to wild-type levels by a second , compensatory mutation in the 3' UTR. These observations are consiste nt with a higher-order structure model that includes long-range intera ctions between the 5' and 3' ends of the Adh mRNA. However, our result s do not fit the classical compensatory substitution model because the second mutation by itself (in the 3' UTR) did not show a measurable r eduction in gene expression.