Gene expression is regulated by auxiliary factors in both a positive and negative manner to increase or decrease expression of specific genes. This regulation often involves direct interaction of factors with the transcription complex to modulate different stages in the transcription process. This proposal addresses the mechanism of transcriptional antitermination of the ribosomal RNA (rRNA) operons of E. coli responsible for the prevention of transcriptional polarity of these genes. Several E. coli proteins (Nus factors) and specific sequences in the DNA have been identified that are involved in modification of transcription complexes initiated from the rRNA promoters. It has been proposed that the antitermination transcription complex is assembled via direct and specific interactions between one or more of these Nus factors, and the nascent RNA complementary to these antitermination sequences in the DNA (boxA and boxB sequences). Photochemical crosslinking will be used to test this model and identity the nature of specific protein-RNA interactions. A new series of nucleotide analogs that contain photoreactive crosslinking groups on the base in positions that do not interfere with normal basepairing will be incorporated in vitro into the nascent rRNA. Active transcription complexes will then be irradiated with long wavelength ultraviolet light to cause covalent attachment of the modified RNA to adjacent proteins, and specific interactions with the transcription factors and RNA polymerase will be examined. Protein-RNA interactions will be identified at the level of specific nucleotides in the RNA and domains and/or amino acids in the proteins. Specifically, the following questions will be addressed: 1. Do Nus factors interact directly with the RNA? 2. Do Nus factors alter the interaction between the polymerase itself and the RNA? 3. Does the termination resistent transcription complex retain the RNA encoded by the antitermination sequences? 4. Does the putative antitermination protein in E. coli also required for antitermination of the rRNA operons interact with the RNA, and can it be identified by crosslinking?
