The AsiA protein, a novel transcriptional regulator and product of the T4-bacteriophage asiA gene, is lethal to bacterial cells. The natural antibiotic tendencies of this protein, combined with the fact that it interacts with highly conserved elements of the machinery in cells - the RNA polymerase complex - central to gene transcription and regulation of gene transcription, suggests that novel, broad-spectrum antibiotics can be developed based on AsiA and the mechanism by which it functions. Because of the increasing resistance of pathogenic bacteria to available antibiotics, new antibiotics and strategies for antibiotic development are critical for fighting bacterial infections and maintaining the public health. The long-term objective of our research is to understand the mechanistic and structural details of transcription regulation. Our immediate focus is on transcription initiation in bacteria and intermolecular recognition and interaction events that regulate the initiation process. Accordingly, the AsiA protein is of significant interest. AsiA is a unique molecular switch that dictates promoter selection during transcription of the phage genome by the host - E. coli - RNA polymerase and is one of the principal factors governing the direction and progression of transcription of the T4 genome. AsiA binds tightly to the sigma70 subunit of the RNA polymerase holoenzyme. This tight interaction was thought to be wholly responsible for its function. However, we have recently discovered that AsiA also binds to other components of the RNA polymerase core and appears also to recognize DNA via a helix-turn-helix DNA binding motif. These attributes form the foundation of our overall hypothesis that AsiA and its orthologs are unique molecular adapters that mediate the structural reorganization of the transcription initiation complex and promoter recognition and selection via interactions with sigma70, the beta subunit, and, under some circumstances, also the promoter DNA.
Our aims are (1) to examine the structural and functional consequences of the AsiA-beta subunit interactions, (2) to define the mechanism of transcription regulation by a family of proteins related to AsiA, and (3) to determine how AsiA-DNA interactions contribute to transcription regulation by AsiA. We will build high resolution structural models of the proteins and protein complexes using NMR, combine mutational analyses with surface plasmon resonance to determine affinities and free energy contributions of individual amino acids to complex formation, and use chemical cross-linking and in vitro transcription methods to characterize interactions with DNA. Overall, our results will provide an intricate structural and functional account of transcription regulation by AsiA and related proteins, will provide new insight into the realms of protein structure and protein interactions in general, and will provide the critical chemical view necessary for pursuit of drug design to mimic the antibiotic properties of AsiA.
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