CHARACTERIZATION OF THE HYPOXIA-INDUCIBLE PROTEIN-BINDING SITE WITHINTHE PYRIMIDINE-RICH TRACT IN THE 3'-UNTRANSLATED REGION OF THE TYROSINE-HYDROXYLASE MESSENGER-RNA

Reduced tension of O-2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis , in the pheochromocytoma (PC12) clonal cell line, The observed increa se in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich t ract located between bases 1552-1578 in the S'-untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)), The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mut ational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypo xia-inducible protein binding site that is represented by the motif (U /C)(C/U)CCCU, where the core binding site is indicated by the underlin ed cytidines, Substitutions of either one of the cytidines with purine or uridine abolished the protein binding, The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of prote in binding for extracts from hypoxic cells. The hypoxia-induced increa se in complex formation was proportional to the strength of binding us ing proteins from normoxic cells, The HIPBS element is conserved in TH mRNAs derived from different species.