STRUCTURAL ANALYSIS OF BIOLOGICAL MACROMOLECULES

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Research will proceed on the molecular basis of genetic control with primary focus on the mechanisms of transcriptional regulation and the chemistry of specific protein/DNA interactions. We will continue to use high resolution X-ray crystallography to establish the structure(s) of the individual macromolecular partners and their specific complexes. The inferences drawn from these structures will be challenged by biochemical and genetic experiments. Finally, a quantitative physical chemical description of the interaction both time averaged and dynamic, will be developed to explain the biochemistry that targets regulatory proteins to their cognate DNA elements. The prokaryotic systems to be studied are: (1) the trp repressor/operator system whose structure and chemistry is now understood in great detail and, - (2) the arg repressor/operator/resolvase system. Eukaryotic systems involved in RNA polII regulation will be studied including: (1) the steroid receptor/response element, (2) B-zip proteins /target DNA, (3) E2/enhancer of bovine papillomavirus, (4) ACE- l/UASc, a metalresponsive transcription factor and its DNA target, (5) TFIID/TATA-box, a key member of the core polymerase complex that fixes the position and polarity of transcription. Cocrystals exist for all of the above systems except TFIID/TATA-box complex. Our longer range focus is to study the interacting elements of the PolII regulatory assembly. This will require the engineering of compact and well defined multimeric protein/DNA complexes suitable for crystallography. The differentiation, growth and metabolism of healthy cells depends on the precise regulation of gene expression. To the extent that failures or imbalances of transcriptonal control play a role in such abnormalities as viral infection, malignant transformation, and endocrine disorders the studies proposed here are of fundamental biomedical importance.