Application): Gene expression by RNA polymerase II activity is regulated by a set of proteins (transcription factors) that determine which genes are to be transcribed. The best characterized of the transcription factors are the helix-loop-helix (HLH) proteins. The HLH proteins typically consist of a basic amino acid sequence,, a helix-loop-helix domain, and a leucine heptad repeat or leucine zipper. Even though the general mechanism of action of HLH transcription factor proteins is known, there is little quantitative solution data on how these proteins interact with each other and their DNA targets. The subgroup myc, Max, Mad, and USF often function as transcriptional activators, but myc may require formation of a myc-Max heterodimer to achieve activation. The Mad-Max heterodimer has the ability to bind to the myc consensus sequence and act as a repressor. These proteins regulate a variety of cellular genes, including sometimes acting as oncogenes and causing malignancy. The long-term goal of this research is to understand and causing malignancy. The long-term goal of this research is to understand how these proteins recognize some but not all DNA target sequences; are able to act as repressors in some cases and activators in other cases; and to understand the kinetics and rate-limiting steps for these processes in order to formulate a more detailed mechanism of gene regulation. Fluorescence spectroscopy will be used to determine equilibrium binding affinity and kinetic properties of these proteins interacting with target DNA sequences and each properties of these proteins interacting with target DNA sequences and each other. Structural data will aid in understanding differences in binding affinity for DNA with variation in the flanking sequences of the target E-BOX and the effects of modification of the protein. The PI will determine if repressor complexes bind more tightly to DNA and block transcription factors and do not bind to DNA. Using USF as an example of this class of proteins, a determination of a more detailed kinetic mechanism of transcriptional regulation will be formulated.
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