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theses

The antibiotics lipiarmycin (Lpm), GE23077 (GE), and salinamide (Sal) function by inhibiting bacterial RNA polymerase (RNAP). In this work, the targets (and mechanisms) of transcription inhibition by Lpm, GE, and Sal are identified and characterized through a combination of genetic, biochemical, and structural approaches. Each of these compounds functions through a different target on the enzyme that does not significantly overlap the targets of other bacterial RNAP inhibitors. Elucidation of these targets may prove useful for antibacterial drug discovery and design. To define the functional target of Lpm, we isolated and sequenced 160 Lpm-resistant mutants. In the structure of RNAP, sites of substitutions conferring Lpm-resistance cluster to define the “Lpm target,” which includes residues in the RNAP switch-region, as well as one wall of the RNA exit channel. Biochemical experiments show that Lpm inhibits the RNAP-DNA interaction, and appears to function by trapping the RNAP clamp in a fully-to-partially closed state. To define the functional target of GE, we isolated and sequenced 35 GE-resistant mutants. In the structure of RNAP, sites of substitutions conferring GE-resistance cluster to define the “GE target,” which includes residues in the RNAP active-center subregions, the “β D2-loop” and the “link region.” Biochemical experiments reveal that GE inhibits nucleotide addition during transcription initiation, after open complex formation, but prior to phosphodiester bond formation. The crystal structure of RNAP in complex with GE confirms that GE binds to the GE target, and indicates that GE functions by precluding the binding of NTP substrates to the RNAP active-center "i site" and "i+1 site.” To define the functional target of Sal, we isolated and sequenced 47 Sal-resistant mutants. In the structure of RNAP, sites of substitutions conferring Sal-resistance cluster to define the “Sal target,” which includes residues in the RNAP active-center subregions: the “bridge-helix N-terminal hinge” (BH-HN), the “F-loop,” and the “link region.” Biochemical experiments reveal that Sal inhibits nucleotide addition during both transcription initiation and elongation. The crystal structure of RNAP in complex with Sal confirms that Sal binds to the Sal target, and suggests that Sal functions by trapping the BH-HN in a straight (unbent) conformation.

ETD_5285

Ph.D.

Includes bibliographical references

by David D. Degen

doi:10.7282/T3125QRJ

ETD doctoral

The author owns the copyright to this work.