Identification of essential nucleotides in an upstream repressing sequence of Saccharomyces cerevisiae by selection for increased expression of TRK2

The TRK2 gene in Saccharomyces cerevisiae encodes a membrane protein involved in potassium transport and is expressed at extremely low levels. Dominant cis-acting mutations (TRK2(D)) selected by their ability to confer TRK2-dependent growth on low-potassium medium, identified an upstream repressor element (URS1-TRK2) in the TRK2 promoter. The URS1-TRK2 sequence (5'-AGCCGCACG-3') shares six nucleotides with the ubiquitous URS1 element (5'-AGCCGCCGA-3'), and the protein species binding URS1-CARl (URSF) is capable of binding URS1-TRK2 in vitro. Sequence analysis of 17 independent repression-defective TRK2(D) mutations identified three adjacent nucleotides essential for URS1-mediated repression in vivo. Our results suggest a role for context effects with regard to URS1-related sequences: several mutant alleles of the URS1 element previously reported to have little or no effect when analyzed within the context of a heterologous promoter (CYC1) [Luche, R. M., Sumrada, R. and Cooper, T. G. (1990) Mol. Cell. Biol. 10, 3884-3895] have major effects on repression in the context of their native promoters (TRK2 and CAR1). TRK2(D) mutations that abolish repression also reveal upstream activating sequence activity either within or adjacent to URS1. Additivity between TRK2(D) and sin3Δ mutations suggest that SIN3-mediated repression is independent of that mediated by URS1.