FUNCTIONAL-CHARACTERIZATION OF AN INOSITOL-SENSITIVE UPSTREAM ACTIVATION SEQUENCE IN YEAST - A CIS-REGULATORY ELEMENT RESPONSIBLE FOR INOSITOL-CHOLINE MEDIATED REGULATION OF PHOSPHOLIPID BIOSYNTHESIS

A repeated element, the inositol-sensitive upstream activation sequenc e (UAS(INO)), having the consensus sequence, 5'-CATGTGAAAT-3', is pres ent in the promoters of genes encoding enzymes of phospholipid biosynt hesis that are regulated in response to the phospholipid precursors, i nositol and choline. None of the naturally occurring variants of the U AS(INO) element exactly recapitulates the consensus (for review, see C arman, G. M., and Henry, S. A. (1989) Annu. Rev. Biochem. 58, 635-669 and Paltauf, F., Kolwhein, S., and Henry, S. A. (1992) in Molecular Bi ology of the Yeast Saccharomyces cerevisiae (Broach, J., Jones, E., an d Pringle, J., eds) Vol. 2, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The first six bases of the UAS(INO) element are homologo us with canonical binding motif for proteins of the basic helix-loop h elix (bHLH) family. Two bHLH regulatory proteins, Ino2p and Ino4p from yeast, were previously shown to bind to promoter fragments containing this element. In the present study, an extensive analysis of UAS(INO) function has been conducted. We report that any base substitution wit hin the putative bHLH binding site resulted either in a dramatic reduc tion or in a complete obliteration of UAS(INO) function as tested in a n expression assay in vivo. Base substitutions in the 5' region that f lanks the 10-base pair repeat, as well as sequences within the repeat itself at its 3' end outside the bHLH core, were also assessed. The tw o bases immediately flanking the 5' end of the element proved to be ve ry important to its function as a UAS element as did the two bases imm ediately 3' of the bHLH core motif. Substitutions of the final two bas es of the original ten base pair consensus (i.e. 5'-CATGTGAAAT-3') had less dramatic effects. We also tested a subset of the altered element s for their ability to serve as competitors in an assay of Ino2p . Ino 4p binding. The strength of any given sequence as a UAS(INO) element, as assayed in vivo, was strongly correlated with its strength as a com petitor for Ino2p . Ino4p binding. We also tested a subset of the modi fied UAS(INO) elements for their effects on expression in vivo in a st rain carrying an opi1 mutation. The opi1 mutation renders the coregula ted enzymes of phospholipid synthesis constitutive in the presence of phospholipid precursors. All elements that retained some residual UAS( INO) activity when tested in the wild-type strain were constitutively expressed at a level comparable with the wild-type derepressed level w hen tested in the opi1 mutant. Thus, UAS(INO) appears to be responsibl e for OPI1 mediated repression, as well as Ino2p . Ino4p binding. Furt hermore, each of the identified functions of the UAS(INO) element appe ars to have the same sequence specificity, and all require the presenc e of the intact bHLH motif, suggesting that transcriptional activation , repression, and Ino2p . Ino4p binding are all components of a single regulatory mechanism.