The title of this application is Transcription Activation by CAP, but in fact, the research proposed covers a much wider range of topics related to transcription initiation. Dr. Ebright also has a grant for work on the alpha subunit of RNA polymerase and for study of eukaryotic transcription. Four basic objectives are described, the first of which does directly involve CAP. In the activation of promoters like lac, a particular region of CAP, called AR1 interacts with the alpha subunit of RNAP. Dr. Ebright wishes to randomize the amino acids of AR1 and select those that are as active or more active than wild type. A second major objective of the work is the study of the alpha subunit of RNAP. First, the residues of alpha that become protected from hydroxyl-radical attack by CAP will be determined. Next, because at least the N-terminal domain of alpha possesses a dimeric structure, but the two subunits do not interact with beta and beta prime symmetrically, the two alpha subunits can be distinguished. Using techniques similar to those used previously for the generation of asymmetric dimers, Dr. Ebright will generate asymmetric RNAP and determine which of the two distinguishable subunits of alpha interacts with CAP, promoter upstream elements, with other activators and repressors. The next class of experiments Dr. Ebright proposes is cross linking. Dr. Ebright has already developed and is now doing experiments in which a radioactive photoactivatable cross linking moiety is placed at any desired nucleotide in a promoter region. Then, anytime during the initiation process, the tag can be activated to link to whatever protein is nearby. Not only can the nearby protein be identified, but the labeled protein can be fragmented so as to allow determination of the labeled peptide within the protein. It is proposed that this type of experiment will be done with the linker placed on every other phosphate of both DNA strands for several promoters, both CAP dependent and independent. In addition to DNA-protein cross linking, protein-protein cross linking experiments will also be performed. The objective of these will be to determine whether there is a specific order of formation and breakage of contacts between CAP and RNAP and DNA in the process of transcription initiation and what it is. Dr. Ebright has developed and already applied the necessary cross linking technology for these experiments. The final line of experiments proposed by Dr. Ebright is the synthesis of a functional activating region epitope. Peptides of constrained conformation due to their cyclization will be used in addition to any sequences found from the first objective of finding superactivators. Randomized sequences will also be used in the goal of finding a short peptide that will substitute for CAP in either occupying alpha and thereby blocking CAP function at a promoter, or in assisting RNAP binding if the peptide is fastened to the DNA in the region where CAP would normally bind.
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