The proposed work is a continuation of previous studies on the regulation of three processes in E. coli: 1. Enzyme formation in the biosynthetic pathway of arginine, controlled by the arginine repressor. 2. Initiation of plasmid replication and maintenance of plasmid stability; the latter process is also controlled by the arginine repressor. 3. Synthesis of the heat-labile enterotoxin, LT. Toward the first project, we plan to elucidate the structure of the repressor molecule, using mutants and fragments of the repressor protein to determine binding sites for arginine and DNA and for the oligomerization site involved in hexamer formation of the repressor. We shall also use calorimetric analysis for the binding of arginine and of DNA to the repressor. In addition, we plan to study how the repressor functions in controlling levels of enzyme synthesis within the arginine regulon. For this, we plan to study the contribution of each of the two promoters of argR, the gene for the repressor. One of the promoters is autoregulated, the other is not. For the second project, we plan to study the interaction of the RepA1 protein, which is required for initiation of replication of replicon RepFIC, with its origin DNA. We shall define the minimal origin region by generating deletions and we shall then study binding to the origin by in vivo footprinting experiments. For studying the effect of the arginine repressor on plasmid stability, we shall delete the ARG box present in the region of RepFIC which is involved in stable maintenance. The arginine repressor has been shown to function in the resolution of plasmid multimers back to monomers. Formation of multimers causes instability. For the third project, we plan to study the effect of global regulatory proteins, such as the product of the lrp gene and the integration host factor (IHF) on the formation of LT. For this purpose we shall study LT formation in mutants unable to produce these regulatory proteins. We also plan to study the temperature control of toxin synthesis, using mutants in which temperature control is altered.
| Beyder, Arthur; Sachs, Frederick (2006) Microfabricated torsion levers optimized for low force and high-frequency operation in fluids. Ultramicroscopy 106:838-46 |
