CAREER: The Influence of ppGpp on the Cellular Actions of BipA

University Of Connecticut, Storrs CT

Investigators

Abstract

The selective synthesis of proteins is crucial for the ability of microorganisms to adapt to, and subsequently overcome, adverse environmental conditions. Exactly how protein biosynthesis is coupled to stress responses and virulence pathways in bacteria is not understood. BipA is a member of the translational family of GTPases and has been shown to be critical to the regulation of stress adaptation and pathogenic processes in microorganisms. Similar to other bacterial GTPases, the properties of this protein are determined by guanine nucleotide binding and exchange, as well as by association with the ribosome. An examination of the ribosome binding properties of BipA reveal an unanticipated linkage between BipA and guanosine tetraphosphate (ppGpp), an alarmone responsible for adaptation to altered growth conditions in bacterial cells. Moreover, evidence suggests that BipA acts as an intermediary between the ribosome and the cellular environment and that its structural and regulatory properties influence how the ribosome responds to changing cellular conditions. This research involves using biochemical and biophysical techniques to understand how BipA accomplishes these tasks at the molecular level. There are at least two BipA ribosome complexes, 70S:BipA:GTP and 30S:BipA:ppGpp that exist in vivo. The first goal of this project is to use protein crystallography, cryo-electron microscopy (cryo-EM) and other biophysical methods to determine the nature of these complexes, map the interaction surfaces between the various components and assess conformational changes resulting from binding and hydrolysis events on the local and global level. The second aim utilizes site directed mutagenesis, deletion analysis, fluorescence and hydrolysis assays to assess the contributions of intra- and inter-molecular interactions to the regulation and activities of BipA. Lastly, a variety of techniques will be employed to understand the biochemical and physiological consequences of the interactions involving ppGpp, BipA and the ribosome. Taken together, these studies will define BipA's molecular mechanism of action and provide a greater understanding of stress and virulence pathways in bacteria. Broader Impact: This project has the potential to transform our understanding of basic processes regulating protein synthesis, a fundamental process crucial to the ability of organisms to grow and respond to their environment. In addition, this CAREER award will support research and teaching activities at the intersection of biochemistry, biophysics, molecular genetics and microbiology. Emphasis has been placed on connecting biophysical techniques and microbiological processes especially in affording cross-disciplinary research opportunities for students at various stages of their educational careers. In addition, laboratory modules will be developed to introduce students to fundamental techniques in biophysics. These adaptable modules provide the basis of outreach programs connecting with local colleges and minority students.