Molecular Mechanisms Of Cu-responsive MAC1 Gene Transcription Regulation

Copper is indispensible for a number of physiological processes. When present in excess, copper is toxic to living organisms. Therefore, livinging organisms must maintain copper at a level that is not too low and not too high. As a copper-sensing transcription factor, Macl plays a critical role in yeast copper homeostasis. Macl regulates transcription of the genes critical to the high-affinity copper transporting machinery such as CTR1, which is central to copper homeostasis in the yeast Saccharomyces cerevisiae. Currently, very little is known about how MAC1 gene transcription itself is regulated. Determining how MAC1 gene is regulated will help us to understand the molecular mechanism of copper homeostasis.We investigated the transcription regulation of MAC1. We found that MAC1 promoter lacks functional TATA-box and Inr elements. Surprisingly, we found that when MAC1 promoter was truncated down to 5 basepair relative to ATG codon of MAC1, the mutant allele of MAC1 was still fully functional as judged by its ability to enable yeast cells to grow normally on copper-demanding growth medium such as YPE. This unexpected result indicates that the MAC1 promoter is likely located within MAC1 open reading frame. The transcription of MAC1 gene is therefore regulated by a novel mechanism that is yet to be fully elucidated.We also studied whether or not the Cu2+ -dependent Cu,ZnSODl anti-oxidant enzyme affects the expression of the well-known heat shock protein encoded by HSP104. Work in the laboratory has discovered that Cu2+ acted as cofactor of Cu,ZnSOD1 is essential to yeast acquisition of thermo tolerance. We found that comparing to the wild type cells, HSP104-lacZ expression was enhanced in the sodlΔmutant yeast cells under both heat shock and non-heat shock conditions. We also found that treating the sodlΔmutant cells with N-acetyl cysteine, a known free radical scavenger agent, was able to reduce the HSP104-lacZ expression but not to the levels seen in the wild type cells. These results suggest that the loss of defense against oxidative stress is probably not the only cause leading to abnormally high expression of heat shock protein expression of HSP104 seen in the sodlΔmutant.