The URE2 IRES of yeast: physical characterization and regulation by eIF2A

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Previous studies established that the cellular URE2 mRNA contains an IRES element in the region encoding the N‐terminus of Ure2p. We have determined the minimal IRES element for this mRNA. It is a stem of about 10 base pairs and with a loop of about 24 nucleotides. The size of the loop is a determinant of efficiency with smaller loops leading to reduced levels of expression. To date, all mutations that increase the stability of the stem reduce the level of expression. At the same time, limited mutations that destabilize the stem also show reduced expression. Thus, it would appear that an optimal balance of stability has been selected naturally in yeast.All of the IRES constructs tested are effectively repressed by eIF2A. We conclude that the regulation by eIF2A is not due to any cis‐acting element in the mRNA, but rather to the process of internal initiation. We are currently attempting to determine if there are trans‐acting factors that influence the expression from the URE2 IRES element or other portions of the URE2 mRNA that enhance or repress IRES‐mediated expression. The regulation of eIF2A is also being examined. Studies of the eIF2A mRNA indicate that most/all stresses lead to a dramatic reduction in eIF2A mRNA levels. However, we do not see uniform reductions in eIF2A protein and are currently evaluating whether post‐translational modifications may be associated with those circumstances. (NIH GM068079 and T32 GM08056)