Structural approaches to eukaryotic transcription

The two projects included in this dissertation explore the interactions of transcription factors with specific binding sites on DNA as well as those protein-protein contacts formed between factors bound to adjacent sites on DNA in transcriptionally active complexes.; The first project presents the crystal structure of a binding specificity mutant of the yeast transcriptional activator GCN4 bound to DNA. By replacing certain residues in the DNA-binding domain of GCN4 with the corresponding ones from the mammalian transcription factor C/EBP, the binding specificity of the protein is converted from recognizing AP-1 and ATF/CREB sites, TGA(C/G)TCA and TGACGTCA respectively, to recognizing the C/EBP site, TTGCGCAA, exclusively. The structure of the complex of the mutant, GCBP8, with C/EBP DNA shows that two residues are largely responsible for the change in specificity, Val239 and Arg243. Val239, mutated from Ala239 in GCN4, recognizes the thymine residues of the TTGC halfsite though hydrophobic contacts. Arg243 is invariant in all members of the bZIP family, but in this context undergoes a dramatic rearrangement with respect to GCN4 to make contact with the pair of guanine residues (on opposite strands) at the 5' end of the halfsite.; The second project presents a large-scale model of the HIV-1 LTR core promoter. For the various subtypes of HIV-1, possible interactions between adjacently bound transcription factors are examined. In particular, the L1 loops of the p50 and p65 subunits of NF-kappaB appear to be potential locations of protein-protein contact. The common alignment of the Sp1 molecules in the model led to the observation that the relative positioning of the Sp1 binding sites with respect to the TATA box is also found in a number of other human promoters that involve Sp1. Similar observations were made with respect to NF-kappaB. These correspondences are most likely reflective of physical interactions between the activation domains of these transcription factors and proteins in the TFIID complex.