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dc.contributor.authorCrane, Benjamin Patrick
dc.description.abstractAnti-sigma factor proteins inhibit transcription in bacteria by binding to the sigma () subunit of the RNA polymerase. A unique anti-sigma factor is the T4 bacteriophage AsiA protein. In E. coli, AsiA interacts tightly with the  subunit bound to the polymerase, and, like other anti-sigma factors, inhibits transcriptional activities. Specifically, AsiA inhibits transcription from bacterial and early phage promoters. AsiA has a unique second function. It assists in transcription activation from phage middle promoters. Thus, AsiA is a unique molecular switch for transcriptional regulation. Moreover, excess AsiA kills bacteria, suggesting AsiA could serve as a model for antibiotic development. To date, most structural and physical studies of AsiA have been performed at relatively low pH (6.5-7.0), and results show that AsiA is an all-helical, symmetric homodimer at high concentrations. However, recent data indicates that AsiA undergoes a substantial, pH-dependent structural reorganization across the range of physiological pH of E. coli (7.4-7.6). Using nuclear magnetic resonance (NMR) spectroscopy, we monitored AsiA structure as a function of pH. As the pH increases from 6.5 to 7.8, the spectra indicate increasing structural heterogeneity. Interestingly, as the pH is further raised, the heterogeneity decreases. At pH 8.1, the spectra indicate a stable, monodisperse single species, which is structurally distinct from species at low pH. A comparison of the NMR chemical shifts of the low and high pH forms indicates clear, localized structural changes. Furthermore, preliminary sedimentation equilibrium experiments indicate decreased affinity between the two protomers at high pH. To further characterize the pH-dependent structural changes, the structure of AsiA was determined at pH 8.1 using residual dipolar couplings (RDCs). Our results indicate that an equilibrium mixture of two distinct structural forms of AsiA exists at physiological pH and that pH-dependent AsiA regulation results from functional differences between these forms. Adaptation of the model for antibiotic development to include pH-dependent regulation could improve potential antibiotic efficacy.
dc.subjectanti-sigma factor
dc.subjectnuclear magnetic resonance
dc.subjectresidual dipolar coupling
dc.subjecttranscription regulation
dc.subjectE. coli
dc.titlePhysical and structural basis for pH dependence of transcriptional regulation by the anti-sigma factor AsiA
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorJeffrey Urbauer
dc.description.committeeJeffrey Urbauer

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