The upstream repression sequence from the yeast enolase gene ENO1 is a complex regulatory element that binds multiple trans-acting factors including REB1.

Cis-acting sequences that modulate ENO1 URS (upstream repression site) element activity were identified by base pair substitution mutagenesis. Base substitution mutations within three distinct regions of the 125-base pair URS element caused partial loss of URS activity in vivo. A URS element containing all three mutations was inactive. A binding site for the yeast REB1 protein was identified near the 5' terminus of the ENO1 URS element. Base substitution mutations that disrupted REB1 binding in vitro caused a 30% loss of URS activity in vivo. A second DNA binding activity was identified which also bound near the 5' terminus of the URS element. This latter binding activity was not antigenically related to REB1 nor was binding of this activity affected by base substitution mutations that abolished REB1 binding. Base substitution mutations within a second region of the ENO1 URS element caused a 38% loss of URS activity in vivo. The nucleotide sequence of this latter region is very similar to essential sequences within the URS elements from the yeast CAR1 and SSA1 genes, respectively. Base substitution mutations within a third region near the 3' terminus of the ENO1 URS element caused a 70% loss of URS activity in vivo. These latter sequences bound a partially purified factor that was distinct from REB1. These results showed that ENO1 URS element activity was modulated by multiple cis-acting sequences that bound distinct trans-acting factors.