Carbon-Detected NMR Studies of Intrinsically Disordered Proteins in Promoter Proximal Pausing

Access to the genetic information encoded in DNA must be both rapid and under tight control so that a cell can readily adapt to its environment, or rapidly respond to growth signals. One regulatory mechanism that is used in nature is to poise the protein machines that access genetic information at the start of genes, waiting to initiate the process of converting the encoded information into molecules that execute cellular functions. Chemical modifications of these ready-to-go proteins exert timely control over cellular responses to stimuli by dictating the efficiency with which genes are activated. For this project, the investigator will push the boundaries of current technologies to explore the structural responses to chemical modifications that trigger release from pausing and entry into efficient decoding of genetic information. This program addresses a significant gap in our knowledge of how gene expression is regulated. This program will train junior scientists at multiple education levels to seek fundamental insight into the systems and processes from biochemistry, using the principles and quantitative laws from the physical sciences. Among other outreach efforts, the investigator will bring undergraduate students from different schools to Penn State for research opportunities that will enhance their career potential in science and engineering fields. To achieve this project’s objectives, new experimental methodology will be implemented for nuclear magnetic resonance (NMR) spectroscopy of highly flexible proteins. While protons are relatively easy to observe by NMR, the PI has shown that the comparatively more challenging direct-detection of carbon yields more quantitative and complete information for intrinsically disordered polypeptides. Recently, the PI used this technique to demonstrate how serine phosphorylation of RNA polymerase II provides a structural switch that plays a role in regulating the critically important enzyme. RNA Polymerase II pauses at the start of genes before entering into productive elongation and these phosphorylation events are associated with release from the paused state. The PI will now broaden the characterization of pause release by investigating threonine phosphorylation on a required co-regulator of RNA polymerase II, named the DSIF complex, which is a second necessary step for pause release. The phosphorylation patterns on RNA Polymerase II change throughout the process of RNA elongation, so this project will extend characterization of serine phosphorylation to model each of the relevant states in an RNA production cycle. Finally, lysine amino acids in a specific region of RNA polymerase II also acquire methyl groups while the enzyme is active on a subset of genes. These modifications can exert combinatorial control with the previously characterized phosphorylation. Therefore, experiments will be performed to test the hypothesis that addition of methyl groups to the amino acid lysine also induces structural transitions, akin to those the PI has observed in association with phosphate incorporation. As a result, this research and associated training activities will yield fundamental, molecular level insights that provide critical molecular insights into gene regulation. This project is funded by the Molecular Biophysics Cluster in the Division of Molecular and Cellular Biosciences. 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.