REGULATORY MECHANISMS OF THE E COLI ARABINOSE OPERON

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The long-term objectives of our work are to understand the mechanisms by which the L-arabinose operons in Escherichia coli are regulated. This will necessitate advances in our understanding of protein structure, DNA structure, protein-DNA interactions, protein-protein interactions, and cell physiology. Activation and repression of transcription by wild type, mutant, and genetically engineered AraC protein will be studied in vivo and in vitro. Tetramerization of AraC protein, as occurs when the protein forms DNA loops, will be studied by gel electrophoresis band shift assays, and an HPLC assay will be further developed and utilized. AraC protein and two structural analogues from the rhamnose operon, RhaS and RhaS, will be studied to determine the amino acids of a subunit that contact the DNA, whether specific amino acids or regions can be identified as activating transcription, why the dimeric AraC protein in some instances contacts three adjacent major grooves of DNA, and in other instances contacts four adjacent major grooves. DNA looping will be investigated. We will test with in vitro transcription whether the same upper and lower limits to loop size exist in vitro as we have found in vivo. Different behavior in vitro would indicate the absence of a protein, perhaps a histone-like protein, that affects looping. By varying the sequences to which AraC protein binds, we will extend observations that the sequence to which AraC protein is bound can affect its behavior. We will also examine the effect on looping and regulation of introducing a stretch of bent sequence into the loop, the effects of changing one AraC protein binding site to another, and of inverting the AraC protein binding sites. We will develop and test a genetic method for locating surface regions of a protein. If it works with aspartate transcarbamoylase, we will apply it to AraC protein and attempt to determine, as well, the regions of the protein involved with activation and tetramerization.