Poster abstracts
Poster number 61 submitted by Elihu Ihms
Structure and Assembly Dynamics of the TRAP-Anti-TRAP Regulatory System
Elihu C. Ihms (Department of Chemistry & Biochemistry, The Ohio State University), Mowei Zhou (Department of Chemistry & Biochemistry, The Ohio State University), Courtney Szyjka (Department of Biological Sciences, State University of New York at Buffalo), Vicki H. Wysocki (Department of Chemistry & Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Mark P. Foster (Department of Chemistry & Biochemistry, The Ohio State University)
Abstract:
In Bacillus spp., the undecameric (11-mer) protein TRAP is activated by elevated intracellular tryptophan concentration, becoming competent to silence several mRNA transcripts implicated in tryptophan biosynthesis. The trimeric protein Anti-TRAP (AT), upregulated during tryptophan starvation, competes for TRAP’s RNA binding sites and restores tryptophan production. Because of their homooligomeric configuration, Both TRAP and AT possess multiple binding sites for their respective partners, raising the possibility that cooperative interactions play an important role in tuning the regulatory circuit.
We investigated this possibility through a battery of bulk structural methods, including sedimentation, small-angle x-ray scattering, and native mass spectrometry. These revealed an unprecedented discovery for regulatory proteins: the polyvalent nature of TRAP and AT enables the formation of irregular, polymeric clusters containing multiple structurally intact copies of both components. This intriguing process suggests a clear mechanism by which small AT trimers compete effectively with the much larger TRAP-RNA interaction surface.
We extended the structural studies by investigating the thermodynamics and kinetics of the TRAP-AT interaction; to deconvolute the data, we engineered AT trimers incompetent to condense TRAP oligomers, and performed stopped-flow FRET experiments to probe the association rates of TRAP+AT and TRAP oligomers. Component binding thermodynamics were investigated by calorimetry, revealing strong negative cooperativity of binding for subsequent AT trimers to TRAP. These experimental parameters were used to build and test a variety of model system behaviors via stochastic simulations, ultimately concluding that TRAP-AT chaining plays an essential role. Consistent with this prediction, RNA competition assays utilizing the chaining-incompetent AT revealed that a higher concentration of this AT was required in order to achieve the same regulatory effect.
References:
Ihms, Elihu C., et al. "Gene regulation by substoichiometric heterocomplex formation of undecameric TRAP and trimeric anti-TRAP." Proceedings of the National Academy of Sciences 111.9 (2014): 3442-3447.
Keywords: gene regulation, reversible oligomerization, hybrid methods