Regulatory single-nucleotide polymorphisms (rSNPs) alter gene expression. Common approaches for identifying rSNPs focus on sequence variants in conserved regions; however, it is unknown what fraction of rSNPs is undetectable using this approach. We present a systematic analysis of gene expression variation at the single-nucleotide level in the Saccharomyces cerevisiae GAL1-10 regulatory region. We exhaustively mutated nearly every base and measured the expression of each variant with a sensitive dual reporter assay. We observed an expression change for 7% (43/582) of the bases in this region, most of which (35/43, 81%) reside in conserved positions. The most dramatic changes were caused by variants that produced AUGs upstream of the translation start (uAUGs), and we sought to understand the consequences and molecular mechanisms underlying this class of mutations. A genome-wide analysis showed that genes with uAUGs display significantly lower mRNA and protein levels than genes without uAUGs. To determine the generality of this mechanism, we introduced uAUGs into S. cerevisiae genes and observed significantly reduced expression in 17/21 instances (p < 0.01), suggesting that uAUGs are functional in a wide variety of sequence contexts. Quantification of mRNA and protein levels for uAUG mutants showed that uAUGs affect both transcription and translation. Expression of uAUG mutants under the upf1Δ strain demonstrated that uAUGs stimulate the nonsense-mediated decay pathway. Our results suggest that uAUGs are potent and widespread regulators of gene expression that act by attenuating both protein and RNA levels.