The research 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, controlled by the RepA1 protein, and 3. synthesis of the heat-labile enterotoxin, LT, controlled by the global regulatory protein H- NS. The amino acid sequences of the three regulatory proteins are known and in one of them the structure has been determined by X-ray crystallography. This facilitates greatly the study of these regulations at the molecular level. For the arginine repressor we plan to study by mutant analysis how the known binding of arginine to the C-terminal domain brings about binding to DNA at the N-terminal domain. We shall also carry out a similar analysis with superrepressor mutants that occur throughout the repressor molecule. For the initiation of RepF1C replication, we shall investigate by in vivo footprinting analysis the role of the RepA1 protein and the state of methylation of the DNA (hemimethylated and fully methylated) in controlling the timing of replication initiation. For the regulation of LT synthesis we plan to investigate the temperature dependence by carrying out in vivo footprinting experiments in the wild type and mutants deleted for H-NS binding sites in the LTA gene.
| Beyder, Arthur; Sachs, Frederick (2006) Microfabricated torsion levers optimized for low force and high-frequency operation in fluids. Ultramicroscopy 106:838-46 |
