DNA-Protein Interactions in Class III Genes

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Proteins which bind to specific base sequences in DNA regulate the expression of many different genes. Transcription factor TFIIIA, the prototype of the Zn-finger class of proteins, binds strongly to a defined base sequence contained within the coding region of 5S RNA genes. This interaction represents the primary step in assembly of a transcriptionally active complex and, once assembled, these complexes persist through many rounds of transcription. The objective of this research is an understanding of the molecular interactions between this protein and its target sequence during complex formation and transcription. To facilitate these studies, a photosensitive cross-linking reagent has been developed, 5-N3-deoxyuridine-5'- triphosphate, which can be enzymatically incorporated into DNA in place of thymidine. Photoactive DNA can be used to label sites or regions in proteins which are in close proximity to the DNA, Contacts between purified TFIIIA and specific positions in the Xenopus. 5S, DNA internal control region have been mapped. This allows examination of the fine structure of this interaction to understand the recognition process, determine how it is affected by the presence of other transcription factors as well as RNA polymerase III, and look for changes which occur as the polymerase initiates RNA synthesis and traverses the TFIIIA contact sites.