Poster abstracts
Poster number 14 submitted by Joe Hazel
Kinetic and Thermodynamic Analysis of the Regulation of the trp RNA-Binding Attenuation Protein (TRAP) By Anti-TRAP
Joseph M. Hazel (Department of Chemistry and Biochemistry, Molecular Biophysics Training Program), Elihu C. Ihms (Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health), Paul Gollnick (Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo), Mark P. Foster (Department of Chemistry and Biochemistry)
Abstract:
Bacterial biosynthesis of tryptophan is regulated by complex protein machinery that is sensitive to several intracellular cues including pH, tryptophan concentration, and levels of uncharged tRNATrp. In Bacillus subtilis regulation involves the trp RNA-binding attenuation protein (TRAP), an 8 kDa protein that forms homo-undecameric rings (TRAP11) in solution. Binding of free tryptophan to TRAP11 leads to increased affinity of the ring for a series of 11 G/UAG repeats in the 5’-untranslated region of the nascent trp mRNA, disrupting an anti-terminator hairpin in the leader region. Control of the trp operon is also moderated by the TRAP-inhibiting protein anti-TRAP (AT), the expression of which is upregulated by the presence of uncharged RNATrp. Translated AT monomers assemble into trimers (AT3), which may bind tryptophan-bound TRAP, preventing the latter from binding to trp mRNA to attenuate tryptophan biosynthesis. As both AT and trp RNA compete for tryptophan-bound TRAP, it is critical to understand the kinetics and thermodynamics of the AT interaction with TRAP to map out the mechanism of tryptophan regulation. Previous studies of AT3 binding TRAP using a homologue from the thermophile Geobacillus stearothermophilus (Bst TRAP) have shown multiple Bsu AT3 can bind a single Bst TRAP11 and that one Bsu AT3 can bind three Bst TRAP11. These experiments show that hetero-oligomerization leads to reversible protein condensation suggesting a possible mechanism for TRAP attenuation; however, binding of Bsu AT3 to Bsu TRAP has not been similarly examined. Here, we explore the thermodynamics and kinetics of Bsu AT3 binding to Bsu TRAP in the presence and absence of tryptophan to elucidate the mechanism of AT-mediated TRAP attenuation. The binding order, affinity, and cooperativity of AT3 binding to TRAP are characterized with isothermal titration calorimetry, fluorescent labeling, and stopped-flow experiments; further clarifying the mechanisms involved in tryptophan biosynthesis regulation.
Keywords: TRAP, Transcriptional regulation, RNA binding