Abstract
H-NS is a nucleoid-associated protein in Escherichia coli that plays a role in DNA\r organization, and acts as a transcriptional repressor. Its ability to form DNA bridges in its\r oligomerized form traps and occludes RNA polymerase preventing transcription of targeted\r genes. To investigate the effect of the functional domains of H-NS on its repression abilities,\r mutations were designed that target each domain independently. H-NS mutations were made by\r site-directed mutagenesis on the pCA24N plasmid and moved onto the chromosome via\r recombination techniques. Assays were conducted to look at the phenotypes of the mutants when\r overexpressed, and the mutants’ abilities to repress. The mutant in the oligomerization domain\r (L33E) is potentially unable to form DNA bridges and is toxic to the cell when constitutively\r overexpressed. A charge-changing mutant in the DNA pocket (R114E) may form weak bridges\r and is able to repress genes. The A117W mutant that alters the shape of the DNA pocket is able\r to form DNA bridges and is like wild type in other assays. To continue describing the mutant\r phenotypes, biochemical and single molecule experiments can be used, allowing the mutants to\r be used to investigate the importance of DNA binding versus oligomerization for the role of HNS\r in various processes.