The heat shock factor (HSF) is a pivotal transcriptional factor that regulates the expression of genes encoding heat shock proteins (HSPs) via heat shock elements (HSEs). nGAAnnTTCnnGAAn functions as the minimum consensus HSE (cHSE) in vivo. Here we show that the expression of Saccharomyces cerevisiae MDJ1 encoding a mitochondrial DnaJ homolog is regulated by HSF via a novel non-consensus HSE (ncHSE MDJ1 ), which consists of three separated pentameric nGAAn motifs, nTTCn-(11 bp)-nGAAn-(5 bp)-nGAAn. This is the first evidence to show that the immediate contact of nGAAn motifs is dispensable for regulation by HSF in vivo. ncH-SE MDJ1 confers different heat shock responses versus cHSE and, unlike cHSE, definitively requires a carboxylterminal activation domain of HSF in the expression. ncHSE MDJ1 -like elements are found in promoter regions of some other DnaJ-related genes. The highly conserved HSF/HSE system suggests that similar ncHSEs may be used for the expression of HSP genes in other eukaryotes including humans.All organisms possess a highly conserved system that responds to elevated temperatures by transcriptionally inducing genes encoding heat shock proteins (HSPs) 1 to deal with heat stress. The induction requires heat shock transcription factor (HSF) and cis-heat shock elements (HSEs) (1). HSF has two conserved domains, a helix-turn-helix DNA binding domain and a coiled-coil hydrophobic repeat domain needed for trimer or higher order multimer formation (2, 3). HSFs of the budding yeasts (Saccharomyces cerevisiae and Kluyveromyces lactis) uniquely possess two activation domains in the amino terminus (AAD) and carboxyl terminus (CAD) (4, 5), whereas those of fission yeast (Shizosaccharomyces pombe), Drosophilla melanogaster, and vertebrates have CADs alone. HSF is moderately phosphorylated under non-stress conditions and is further activated by hyperphosphorylation upon heat shock to induce the expression of HSPs (6 -8). In fission yeast, D. melanogaster, and mammals, the binding activity of HSFs to HSEs is dramatically stimulated by heat shock as HSF monomers are converted to trimers (9 -11). In contrast, in the budding yeast, HSF constitutively binds to HSE, but the binding activity seems to increase after heat shock-induced hyperphosphorylation (7,(12)(13)(14)(15)(16).The HSE is composed of several contiguous inverted repeats of the 5-base pair sequence nGAAn (where n is any nucleotide) (1,17,18). The number of the pentameric units in HSE varies, but at least three units are thought to be the minimum required for heat regulation in vivo. Namely, nGAAnnTTCnnGAAn is the minimum consensus HSE (cHSE) (19,20). However, an HSE can tolerate and still function with a 5-bp insertion between two repeating units if the spacing and orientation of the pentameric elements are maintained, e.g. nGAAn-(5-bp)-nGAAn (21). The most characterized endogenous non-consensus HSE (ncHSE) is that of CUP1 in S. cerevisiae, nTTCnnGAAn-(5-bp)-nGAGn denoted ncHSE CUP1 (22)(23)(24). Similar elements regulate HSP82 and HSC82 (12,(25)(...