A new class of DNA binding proteins involved in gene activation and cell differentiation has been identified. It has been proposed that this class of proteins contains a unique structural motif, termed a helix-loop-helix, which is believed to bind selectively to sequences of DNA. Immunoglobulin enhancer binding protein E47 (IEB E47) and the products of lyl1 and tal genes contain a putative helix-loop-helix. The gene encoding IEB E47 lies at the t(1:19) chromosomal breakpoint found in certain acute leukemias and is structurally altered by most of the t(1:19) chromosomal translocations. Likewise lyl-1 and tal genes altered by chromosomal translocations in T cell acute lymphoblastic leukemia. These data imply roles for the rearranged genes and their products as factors in human leukemias. The present proposal outlines studies to help define how this class of proteins containing the putative helix-loop-helix motif is involved in the control of normal and abnormal cellular proliferation. The efforts detailed herein will investigate the helix-loop-helix-DNA interaction with the intent of clarifying the structural origin of gene activation.
The specific aims of this proposal are: 1) chemically synthesize two proteins, 86 and 59 amino acids in length (HLH-E47S, and HLH-E47 respectively), containing the putative helix-loop-helix segment of IEB E47 by a novel fragment coupling methodology, 2) solve the solution conformations of HLH-E47S and HLH-E47 using two-dimensional NMR techniques, 3) determine the aggregation states of HLH-E47S and HLH-E47, 4) determine binding constants and the precise DNA binding location of HLH-E47S and HLH- E47 on the kappaE2 enhancer site by gel mobility shift assays and chemical footprinting, 5) chemically synthesize the lyl-1 protein using a similar peptide fragment coupling methodology, and determine the DNA sequences to which it binds by chemical footprinting.
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