Bacteria use many different proteins to sense and respond to environmental changes, often altering levels of transcription from specific genes to alter protein levels. Prokaryotic regulation is relatively simple compared to eukaryotic, and many components of the molecular machinery have been structurally characterized, including the key enzyme, RNA polymerase. The s54-polymerase transcription system provides a direct coupling of chemical sensing to changes in rates of transcription at specific genes, a process mediated by an ATPase activity in required transcriptional activator proteins. Our studies of these activator proteins have shown how receiving a signal (phosphorylation or ligand binding) leads to conformational changes that activate ATPase activity. The ATPase couples chemical energy from ATP hydrolysis into conformational changes in s54-polymerase that enable transcription initiation. Studies of the s54 subunit are providing insights into the nature of the structural changes. The processes of binding-induced response, and ATP driven conformational changes occur in all organisms and many different contexts, the insights generated in this system will help understand many others as well. Our broad goal is to provide a comprehensive molecular level understanding of the function of transcriptional activators and how they act through s54 polymerase. We will continue to focus on Aquifex aeolicus proteins to develop connections with biochemical function, and to understand regulatory mechanisms. We will extend structural studies of s54, providing data to complete a structure of all but the N-terminal 70 amino acids. We will examine how the N-terminal residues of s54 interact with activator proteins, and study the mechanism by which ATP hydrolysis drives the conformational changes that lead to transcription initiation. Using single molecule manipulation experiments we will investigate the response of s54 to mechanical forces, analogous to that applied by the activators. The s54-transcriptional activator system occurs in most bacteria, and is involved in regulating transcription of some key genes that affect virulence and the ability to change hosts. It does not occur in eukaryotes, and hence could be a target for future drug development. Understanding structural mechanics through the proposed work would greatly aid such an effort. The AAA+ domain of the activators is similar to such domains in many human proteins that help reorganize protein complexes, processes that are generally not well understood. Better understanding of the activator ATPase should provide insights into function of other AAA+ proteins.
Cells constantly sense their environment and respond to changes in it to optimize survival. One important response is altering the level of gene transcription to modulate the concentrations of proteins in the cell. The mechanisms for both sensing signals and responding to them are highly varied to provide the appropriate sensitivity and rate of response required for different types of signals. The experiments we propose will study, at the structural level, how sensing by transcriptional activators is coupled to increasing gene transcription by a specific from of bacterial RNA polymerase (with the s54 subunit) that gives a rapid response and dramatically changes the level of transcription. This work has the overlapping goals of understanding the molecular processes that are involved in sensing chemical signals in and around cells and then altering gene transcription, and understanding how the energy of ATP hydrolysis is converted by the transcriptional activators into conformational changes that modulate polymerase activity. The principles that we learn will provide insight into many other systems. Transcription by the s54 system, which occurs only in bacteria, is used for production of virulence factors and proteins important for host interactions, and our studies may provide ideas for new therapeutic targets to treat infections.
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