NSF Award
2210869
This project will determine how the cellular protease Lon, which degrades various proteins, regulates the transcription of a genetic material known as ribonucleic acid (RNA). Since Lon-mediated protein degradation and RNA transcription are conserved in bacteria and humans, findings generated from this work will provide insights into the workings of a universal process that is important for sustaining life. The PI hypothesizes that the competing interaction between Lon and the physiological binding partner(s) for the same cellular protein dictates this cellular protein's fate and, ultimately, function. The project will test this hypothesis by determining how purified Escherichia coli Lon protease (ELon) affects the assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. In addition, the proposed research will determine the extent to which the assembly of the RNA transcription process protects the lambda N protein from degradation by ELon. This project will integrate research into education. The broader impact of this project lies in promoting science literacy to underprivileged children in Cleveland, Ohio. Trainees participating in this project will learn to teach and communicate scientific concepts to the general public, which will prepare them for an education career in STEM. Undergraduate students and post-doctoral fellows will be trained to conduct research in organic synthesis, molecular cloning, protein purification and labeling, electron microscopy, and enzymology. The trainees will learn to teach and communicate scientific concepts to the general public. This kind of experience will provide a training ground for future educators in STEM.<br/><br/>This project will determine if the Escherichia coli Lon protease (Elon) participates in the regulation of RNA transcription. The project will test the hypothesis that Lon disrupts RNA transcription by selectively degrading the lambda N protein, which is needed for antitermination RNA transcription. A combination of enzyme kinetics, mutagenesis, and electron microscopy imaging approaches will be used to evaluate the impact of Escherichia coli Lon protease (ELon) on the kinetic assembly of the RNA transcription complex constituting the Escherichia coli RNA polymerase (ERNAP), the lambda N protein, the NusA protein, and the nucleic acid containing the nutL sequence. The effect of RNA transcription on Lon-mediated proteolysis of the lambda N protein will also be evaluated. Since Lon is found in most organisms, physiologically relevant mechanistic findings on ELon could be transformed to understand the workings Lon homologs in eukaryotes. The quantitative approach developed for elucidating the mechanism of Lon would benefit the adaptation of the same approach to study other ATP-dependent proteases to reveal mechanistic differences among this family of enzymes.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
