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 i nvolved in potassium transport and is expressed at extremely low level s. 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 ubiqu itous URS1 element (5'-AGCCGCCGA-3'), and the protein species binding URS1-CAR1 (URSF) is capable of binding URS1-TRK2 in vitro. Sequence an alysis of 17 independent repression-defective TRK2(D) mutations identi fied three adjacent nucleotides essential for URS1-mediated repression in vivo. Our results suggest a role for context effects with regard t o URS1-related sequences: several mutant alleles of the URS1 element p reviously reported to have little or no effect when analyzed within th e context of a heterologous promoter (CYC1) [Luche, R.M., Sumrada, R. & Cooper, T.G. (1990) Mol Cell. Biol. 10, 3884-3895] have major effect s on repression in the context of their native promoters (TRK2 and CAR 1). TRK2(D) mutations that abolish repression also reveal upstream act ivating sequence activity either within or adjacent to URS1. Additivit y between TRK2(D) and sin3 Delta mutations suggest that SIN3-mediated repression is independent of that mediated by URS1.