STRUCTURE-FUNCTION CORRELATIONS OF THE INSULIN-LINKED POLYMORPHIC REGION

The insulin minisatellite of the insulin-linked polymorphic region (IL PR), a 14 base-pairs long tandem repeat of: 5'-ACAGGGGTGTGGGG-3' 3'-TG TCCCCACACCCC-5', is located 363 base-pairs upstream of the human insul in gene. A locus for insulin-dependent diabetes mellitus (IDDM) has be en mapped to the ILPR. It has been shown that the ILPR is polymorphic in length and this length polymorphism is also related to the transcri ptional activity of the insulin gene and the susceptibility to IDDM. H ere, we attempt to decipher the role of the ILPR structure in length p olymorphism and transcriptional regulation. We show by gel electrophor esis, circular dichroism (CD) and one and two-dimensional nuclear magn etic resonance spectroscopy (1D/2D NMR) that the G-rich strand of the ILPR adopts an intramolecularly folded hairpin G-quartet structure. A detailed analysis of 1D/2D NMR data of d(G(4)TGTG(4)) and d(G(4)TGTG(4 )ACAG(4)TGTG(4)) enables us to define the nature of chainfolding, the stacking interaction of the G-tetrads in the stem, and the interaction s of the bases in the loops. d(G(4)TGTG(4)ACAG(4)TGTG(4)) happens to b e the smallest unit of the G-rich strand that can form the intramolecu lar hairpin G-quartet structure. For long ILPR sequences, several such hairpin G-quartet structures can be linked in space. Indeed, by an in vitro replication assay, we show the presence of such multiple hairpi n G-quartet structures for the G-rich strand of the ILPR of repeat len gth 6. This observation suggests that the formation of multiple hairpi n G-quartets may explain slippage during replication and the observed length polymorphism. From our high resolution structure, we are able t o identify a set of interactions that are critical for the structure a nd stability of the hairpin G-quartet. Single or double mutations in t he ILPR that destabilize these interactions also lower the transcripti onal activity of the insulin gene. Therefore, the hairpin G-quartet st ructure of the ILPR has a direct correlation with the transcriptional activity of the human insulin gene. (C) 1996 Academic Press Limited