This grant describes a series of genetic and biochemical experiments designed to probe the interaction between DNA binding proteins and their recognition sites in double-stranded DNA. The cI, c2, cro, arc, and mnt repressors of bacteriophage Lambda and P22 will be studied. We will also study the inactivation of the Lambda cI repressor and the P22 c2 repressor by the E. coli RecA protein and by the P22 antirepressor protein. The long range goal of this research is to understand how proteins recognize specific sequences of DNA, and to understand how interaction between DNA binding proteins and other proteins regulates DNA binding. Lambda repressor mutants which are properly folded but which show a reduced affinity for operator DNA will initially be identified. We will purify these mutant proteins and will assay their interaction with operator DNA by methylation protection and nuclease protection techniques. The binding of these mutant repressors to non-operator DNA will also be assayed. Such experiments may allow us to identify specific points of contact between the protein and DNA by differences in protection patterns, and should allow an estimate of the energetic loss resulting from perturbation of specific protein-DNA contacts. Similar experiments will also be performed with Lambda cro protein. Since the three-dimensional structures of the Lambda cro protein and the N-terminal, operator binding domain of Lambda repressor are known, our mutant studies should be interpretable in detailed molecular terms. The arc and mnt repressors have yet to be obtained in pure form. We will purify these repressors and will study their interaction with operator DNA. The affects of these proteins on P22 immunity I transcription in vitro will also be determined. Mutants of Lambda repressor resistant to recA cleavage, P22 antirepressor inactivation, and inactivation by dominant negative repressor mutants will be selected and sequenced. We will use these purified mutant repressors, and proteolytic fragments of wild-type repressor to study the mechanism by which the C-terminal domain of repressor mediates RecA cleavage, antirepressor binding, oligomerization, and binding of heteroimmune repressors.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Massachusetts Institute of Technology
Schools of Arts and Sciences
United States
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Hari, Sanjay B; Grant, Robert A; Sauer, Robert T (2018) Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase. Structure 26:1251-1258.e3
Brown, Breann L; Kardon, Julia R; Sauer, Robert T et al. (2018) Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme. Structure 26:580-589.e4
Amberg-Johnson, Katherine; Hari, Sanjay B; Ganesan, Suresh M et al. (2017) Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens. Elife 6:
Totaro, Kyle A; Barthelme, Dominik; Simpson, Peter T et al. (2017) Rational Design of Selective and Bioactive Inhibitors of the Mycobacterium tuberculosis Proteasome. ACS Infect Dis 3:176-181
Baytshtok, Vladimir; Chen, Jiejin; Glynn, Steven E et al. (2017) Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation. J Biol Chem 292:5695-5704
Olivares, Adrian O; Baker, Tania A; Sauer, Robert T (2016) Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines. Nat Rev Microbiol 14:33-44
Hari, Sanjay B; Sauer, Robert T (2016) The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli. Biochemistry 55:5649-5652
Stein, Benjamin J; Grant, Robert A; Sauer, Robert T et al. (2016) Structural Basis of an N-Degron Adaptor with More Stringent Specificity. Structure 24:232-42
Baytshtok, Vladimir; Fei, Xue; Grant, Robert A et al. (2016) A Structurally Dynamic Region of the HslU Intermediate Domain Controls Protein Degradation and ATP Hydrolysis. Structure 24:1766-1777
Barthelme, Dominik; Sauer, Robert T (2016) Origin and Functional Evolution of the Cdc48/p97/VCP AAA+ Protein Unfolding and Remodeling Machine. J Mol Biol 428:1861-9

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