The activity of an upstream repression sequence (URS element) that med
iates a 20-fold repression of ENO1 expression in cells grown in a medi
um containing glucose was characterized. Sequences that are sufficient
for orientation-dependent ENO1 URS element activity were mapped betwe
en positions -241 and -126 relative to the ENO1 transcriptional initia
tion site. The ENO1 URS element repressed transcription of the yeast C
YC1 gene when positioned between the CYC1 upstream activation sequence
s (UAS elements) and TATAAA boxes. The ENO1 URS element failed to repr
ess transcription of the wild-type yeast enolase gene ENO2; however, e
xpression of an ENO2 gene lacking one of the ENO2 UAS elements was eff
iciently repressed by the ENO1 URS element, suggesting that the URS el
ement interferes with the transcriptional activation by some, but not
all, UAS elements. In contrast to the ENO1 gene, the ENO1 URS element
repressed CYC1 and ENO2 expression in cells grown on glucose or glycer
ol plus lactate. Evidence is presented that the ENO1 URS element also
functions during stationary growth phase.