The expression of genes that are encoded by DNA is essential for sustained life. This project will investigate how basic steps in gene expression occur. The first step in gene expression is transcription: the process in which one strand of DNA in a gene is copied to make an RNA molecule. This requires that many different proteins and the DNA assemble into a complex, such that gene expression can occur accurately, and in the correct cell type at the proper time. This research will study the assembly and disassembly of complexes containing proteins and DNA that are essential for transcription to occur. The research will use experiments that allow single molecules to be observed. Studying molecules one at a time will reveal new insight into the diverse behaviors of proteins and DNA important for controlling gene expression. Importantly, this project will promote the training of graduate and undergraduate students, enable new educational curriculum to be developed, and encourage participation of underrepresented minority and women scientists.
The specific objectives of the project are to investigate mechanisms of steps in early mRNA transcription and to understand the pathways by which the transcriptional activator protein p53 binds to DNA. The experiments take advantage of single molecule fluorescence techniques, which have emerged as essential contributors to revealing the dynamic behavior and heterogeneity of biological complexes, thus providing unique insight into their function. Experiments will investigate the mechanism by which the general transcription factor TFIIB releases from early transcribing human complexes in vitro. Importantly, distinctions will be made between active and inactive complexes, thereby directly relating TFIIB release to activity. Studies will also focus on understanding how p53 binds its DNA response elements, which is important for controlling transcription of genes critical to regulating the cell cycle and programmed cell death. The ability to resolve diversity in protein/DNA complexes will reveal the allowable pathways by which p53 forms different oligomeric complexes on DNA, as well as the kinetics of association and dissociation events in these pathways. How different p53 response elements, as well as specific regions and amino acids in p53, control modes of DNA binding will be determined. The knowledge gained from this research will stimulate new models that will broadly inform future studies of transcriptional regulation, both in vitro and in biological systems.
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.
