Poster abstracts
Poster number 168 submitted by Joseph Hazel
Regulation of tryptophan biosynthesis through the interaction of the trp¬ RNA-binding attenuation protein (TRAP) and its inhibitor Anti-TRAP
Joseph M. Hazel (Department of Chemistry and Biochemistry, Molecular Biophysics Training Program, The Ohio State University), Elihu C. Ihms (Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health), Pall Gollnick (Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Biosynthesis of tryptophan in Bacillus subtilis (Bsu) is regulated by complex macromolecular machinery that is sensitive to several intracellular cues including pH, tryptophan concentration, and levels of uncharged tRNATrp. The trp RNA-binding attenuation protein (TRAP), is an 8 kDa polypeptide that forms homo-undecameric rings (TRAP11) in solution. Binding of free tryptophan to TRAP11 leads to increased affinity for a series of 11 G/UAG triplet repeats located in the 5’-untranslated region of the nascent trp mRNA, disrupting an anti-terminator hairpin in the leader region. Expression of the trp operon is further moderated by the TRAP-inhibiting protein anti-TRAP (AT), which is expressed in B. subtilis in in response to increases in uncharged RNATrp. 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 fully determine the mechanism of tryptophan regulation.
Previous studies of AT3 binding TRAP using a homolog from the thermophile Geobacillus stearothermophilus (Bst TRAP) have shown that multiple Bsu AT3 can bind a single Bst TRAP11 and that one Bsu AT3 can bind multiple Bst TRAP11. These experiments showed that hetero-oligomerization leads to reversible protein condensation suggesting a possible phase-separation mechanism for AT/TRAP attenuation. We explore the thermodynamics and kinetics of Bsu AT3 binding to Bsu TRAP11 to better elucidate the mechanism of AT-mediated TRAP attenuation. The binding order, affinity, and cooperativity of AT3 binding to TRAP are explored using biophysical tools for modeling mechanisms of allosteric regulation. This work deepens our understanding of complex machinery cellular machinery involved in fine tuning gene regulation.
Keywords: RNA-binding protein, circular RNA, thermodynamics