No description available.

Chemistry / Although molecules that bind DNA have the potential to modify gene expression, the reality of targeting DNA in a sequence-specific manner is still a problematic but worthwhile goal. The Iverson lab has been exploring DNA recognition through a motif known as threading polyintercalation based on connecting intercalating naphthalene diimide (NDI) units, which are molecules that insert themselves between DNA base pairs, together with peptide linkers. These polyintercalators interact with both DNA grooves by “threading” or winding through the DNA, like a snake might climb a ladder. Initially, two different bisintercalator modules with altered sequence specificities and different groove binding topologies were discovered and used to inspire the design of a hybrid NDI tetraintercalator. Surprisingly enough, this tetraintercalator bound sequence-specifically with a dissociation half-life of 16 days to its preferred 14 bp site, a record at the time it was reported for a synthetic DNA-binding molecule. The work reported here expands on the capabilities of this modular threading polyintercalation motif. Chapter 2 describes the ability of a new hybrid NDI tetraintercalator, where the bisintercalator modules are connected together in a different way compared to the previously studied tetraintercalator, to subtly discriminate between similar binding sites. Chapter 3 offers a structural understanding, through NMR analysis, for the sequence recognition abilities of this new tetraintercalator. Chapter 4 analyzes the binding abilities of an un-optimized NDI octaintercalator and proposes how to approach the second-generation design of longer polyintercalators. Chapter 5 describes the optimization of the originally designed NDI tetraintercalator by serially lengthening one of the linkers to produce a tetraintercalator with a 57 day dissociation half-life from its 14 bp sequence, a new record for a synthetic DNA-binding molecule. Using the optimized linker in the context of an NDI hexaintercalator allows for binding to a 22 bp designed site, a record for a synthetic non-nucleic acid molecule. Chapter 6 recounts a focused library screening to search for bisintercalators with new sequence specificities. These efforts have laid the groundwork to progress toward studies aimed at understanding how these molecules might function to prevent transcription in a sequence-dependent manner in vivo. / text

Pathogenesis of enterotoxigenic Escherichia coli (ETEC) and Shigella flexneri relies predominantly on members of the AraC/XylS family of transcriptional regulators, Rns (or its homolog, CfaD) and MxiE, respectively. Rns/CfaD regulate the expression of pili, which allow the bacteria to attach to the intestinal epithelium. Better understanding of the role Rns plays in virulence was attained by expanding our knowledge of the Rns regulon, revealing that it functions as an activator of cexE, a previously uncharacterized gene. By in vitro DNase I footprinting two Rns-binding sites were identified upstream of cexEp, both of which are required for full activation of cexE. The amino terminus of CexE also contains a secretory signal peptide that is removed during translocation to the periplasm. Though the function of CexE remains unknown, these studies suggest that CexE is a novel ETEC virulence factor since it is regulated by Rns/CfaD. In Shigella flexneri, the expression of a subset of virulence genes (including, ipaH9.8 and ospE2) is dependent upon the activator MxiE and a cytoplasmic chaperone IpgC. To define the molecular mechanism of transcriptional activation by this chaperone-activator pair, an in vitro pull down assay was performed revealing that MxiE specifically interacts with IpgC in a complex. Additionally, IpgC recognizes three polypeptide regions in MxiE: within MxiE(1-46), MxiE(46-110) and MxiE(196-216). Furthermore, it seems that MxiE and IpgC regulate transcription of ipaH9.8 and ospE2 promoters differently. In the bacterium, the formation of the MxiE-IpgC complex is initially prevented because IpgC is sequestered in individual complexes with effector proteins, IpaB and IpaC. Upon contact with an eukaryotic host cell the effector proteins are secreted, thereby freeing IpgC to form a complex with MxiE and activate the expression of virulence genes. This new characterization of the role of Rns and MxiE in virulence gene regulation in ETEC and S. flexneri, respectively will give new insights into the pathogenesis of the regulators.