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KMID : 0624219990070000050
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Focus on Genetic Science
1999 Volume.7 No. 0 p.50 ~ p.67
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Upstream AUGs Are Negative Regulators of Human ADH5/FDH Gene Expression
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Kwon, Hye-Sook
Lee, Dong-Kee/Hur, Man-Wook
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Abstract
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We investigated the translational regulation of the human ADH5/FDH gene by its 5’ untranslated region (5’UTR). The 5’UTR is GC rich and contains an upstream open reading frame (uORF) with two possible AUG start codons, a structural feature uncommon in mammalian genes. We prepared, by site-directed mutagenesis, a series of ADH5/CAT fusion constructs with A to C mutations at one or both of the upstream translation initiation codons. In Drosophila SL2 cells, mutation of either AUG condon increased reported gene expression two folds, demonstrating an inhibitory role of the upstream AUGs. Using in vitro synthesized, capped mRNAs, we investigated the effect of the upstream AUGs on translation in vitro. We found 3- to 5- fold increases in reporter activity when the upstream AUGs were mutated, demonstrating the strong inhibitory effect of these AUGs on translation. Toeprint analysis demonstrated many stalled ribosomes flanking the upstream AUGs codons and the presence ability of ribosomes to recognize the two upstream AUGs, despite their poor initiation contex. The upstream ORF, presence of secondary structures, and short intercistronic distance (2 bp) between the uORF and man ORF are important translational regulatory elements of ADH5/FDH expression.
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