Transcription of genomic DNA into messenger RNA by RNA polymerase is the first step of gene expression, and is extensively controlled in all living organisms. The goal of this project is to understand the molecular mechanism by which a global transcriptional regulator DksA controls the bacterial response to starvation and stress. During starvation, DksA acts in concert with the alarmone ppGpp and to couple the ribosomal RNA transcription, and thus ribosome production, to the cellular demand for protein synthesis. The mechanism of the DksA action is very unusual: unlike most transcription initiation factors, DksA does not recognize a specific DNA sequence and instead directly binds to RNA polymerase near the enzyme's active site to alter the structure and the stability of transcription complexes. In this work, the Artsimovitch laboratory will dissect DksA interactions with RNA polymerase, investigate DksA effects on transcription elongation and termination, and test whether DksA alters the fidelity of RNA synthesis. Since DksA appears to exert its control near the RNA polymerase active site, whose structure and basic mechanism are universally conserved in all domains of life, the paradigms established by this analysis will have far-reaching implications. The broader impact of this project lies in its education and dissemination emphases. The activities will contribute to the training of graduate and undergraduate students who will receive broad multi-disciplinary training in structure/function analysis of a transcription regulator. The project will also generate tools for a laboratory course in microbial genetics and resource materials for other research groups around the world.
The major goal of this study was to characterize the mechanism of DksA, a bacterial transcription factor with unusual properties. Our previous data demonstrated that Escherichia coli DksA binds near the active site of RNA polymerase, an enzyme that catalyzes the synthesis of all RNA messages in bacteria, and alters its catalytic properties. Proteins from the DksA family regulate expression of a large number of genes that are required for response to environmental stresses and for successful execution of virulence programs. Studies from other groups show that DksA can activate or inhibit transcription initiation but, in contrast to most initiation factors, DksA does not interact with the promoter DNA. It was thus not clear how DksA promoter specificity is determined. We used a combination of genetic, biochemical, and structural approaches to study the molecular details of DksA mechanism. First, we showed that DksA action is not limited to initiation of transcription, and that DksA also regulates the subsequent elongation and termination phases. Second, we identified the binding site for DksA on RNA polymerase. Third, we showed that DksA does not compete with a structurally similar GreB protein, which we previously showed to bind to the same site. Our experimental data, together with a molecular docking model, suggest that two domains of RNA polymerase hinder DksA binding, restricting DksA action to transcription complexes in which these domains are mobile due to the lack of stable interactions with the promoter DNA. In summary, we propose a model in which binding of DksA and GreB to the transcription complexes is controlled by the conformation of RNA polymerase, with each factor being able to bind to its target complexes irrespective of the presence of the competing protein(s). Finally, we demonstrated that an essential domain in the promoter-specificity sigma subunit of RNA polymerase is necessary for the regulation by DksA. This result sheds light on the hitherto unknown role of this enigmatic domain in regulation of gene expression in the cell. The second goal of this work was to study DksA2, a DksA homolog from Pseudomonas aeruginosa that also encodes a canonical DksA. DksA2 lacks the zinc finger, the key structural element proposed to be essential for the structural integrity of DksA. We obtained a structure of DksA2 and showed that, quite remarkably, the two proteins have very similar structural folds, despite the absence of the stabilizing zinc atom in DksA2. Our analysis demonstrates that DksA2, which is expressed only under zinc-limiting conditions, binds to the same site on RNA polymerase and can substitute for DksA in vivo and in vitro. These results suggest that DksA2 serves as a backup system for zinc starvation when the primary DksA is unable to function due to the loss of zinc, a condition which bacterial cells encounter upon their entry into the host. Apparently, DksA2 evolved to have an appropriate sequence to be zinc independent and yet to maintain the structure and function of DksA. We suggest that similar structural adaptation may have occurred in other zinc-independent paralogs, such as several bacterial ribosomal proteins. This project was carried out primarily by five graduate and undergraduate students, who have learned and developed new techniques, participated in manuscript preparation, and gave presentations at local and international conferences. Two of the graduate students have successfully defended their Ph.D.s and the undergraduate student began his graduate studies.
