The yeast heme responsive transcriptional
activator HAP1 serves as a paradigm for studying heme signaling in
eukaryotic cells. HAP1 contains a DNA-binding domain, an activation
domain and two heme regulatory domains. The HAP1 DNA-binding domain
encompasses a C6 zinc cluster motif and a coiled-coil dimerization
element typical of the yeast GAL4 family proteins. The heme regulatory
domains--the heme domain and the HRM7 domain--contain respectively six
and one heme responsive motifs (HRMs) that can bind directly to heme. We
have carried out a systematic functional analysis of HAP1 elements
required for heme regulation and transcriptional activation.
Surprisingly, we found that the HRMs are not responsible for heme
regulation of HAP1: HAP1 derivatives with the HRMs in the heme domain or
all HRMs deleted are still repressed in the absence of heme and require
heme for activation. Rather, three novel elements located at the end of
the dimerization domain, in the beginning of the heme domain, and in the
HRM7 domain are critical for heme regulation of HAP1. Further, we found
that the coiled-coil dimerization element is dispensable for DNA binding
and transcriptional activation at UAS1/CYC1, but is essential for
transcriptional activation at UAS/CYC7. The HAP1 dimerization domain
contains multiple elements that can substitute for each other in DNA
binding and transcriptional activation. These unexpected findings
provide novel insights into the mechanism governing heme activation of
HAP1 and transcriptional activation by HAP1.
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