Date of Award
protein alleles, conditional protein splicing
The use of small molecule modulators of protein function (drugs) has gained in popularity due to the speed and precision with which they can work. Unfortunately, a small molecule with the required specificity and potency is not always available. We have developed two methods to generate small molecule sensitive protein alleles without the need to screen for a new drug. In the first, which we term conditional protein splicing (CPS), an intein is split into two inactive fragments which are activated by heterodimerization. By fragmenting the target protein and fusing the pieces to this split intein, we were able to control the target proteinâ€™s activity through splicing. In the second, which we term Split Ubiquitin for the Rescue of protein Function (SURF), a protein is expressed with a destabilizing N-terminal sequence (degron) that leads to its degradation. The degron is removed through heterodimerization induced ubiquitin complementation and cleavage. This stabilizes the protein and allows a build up of activity. These techniques should allow the manipulation of a variety of proteins for the study of biological processes. The bacterial RNA polymerase Ïƒ factors are required for promoter specific transcription initiation. The Ïƒ factors responsible for driving most gene expression during log phase growth, known as the group 1 Ïƒ family are autoregulated by a poorly understood mechanism. Group 1 Ïƒ factors are unable to bind to DNA in the absence of core RNA polymerase. The N-terminal domain of group 1 Ïƒ factors, known as region 1.1 is responsible for the auto-inhibition of DNA binding capabilities. However, since in all crystal structures of bacterial RNA polymerase, region 1.1 is either absent or not resolved, it is not clear how region 1.1 works. We used NMR to determine the structure of region 1.1 and intramolecular crosslinking to provide evidence that region 1.1 acts by directly binding to the DNA binding domains of group 1 Ïƒ factors.
Schwartz, Edmund Ching, "Conditional Protein Alleles and Regulation of Bacterial Transcription: A Thesis in Two Acts" (2008). Student Theses and Dissertations. 4.