This proposal focuses on two phenomena of widespread importance in biology: (1) Allostery in regulation of homo-oligomeric proteins. Homo-oligomeric proteins are widespread and over-represented in regulatory systems. Allosteric communication between ligand binding sites is critical for effective regulation, and understanding mechanisms of allosteric coupling between the subunits (protomers) of homo-oligomers is critical for manipulating biological regulation, and as a means of enabling targeted drug design. However, despite decades of study of allosteric phenomena, the atomic-level linkages between dynamics, thermodynamics and structure remain enigmatic. (2) How proteins remodel noncoding RNA to regulate their function. Proteins play critical roles in proper folding and assembly of structured RNAs, enabling functions that include ribosomal assembly, RNA catalysis and transcriptional regulation. The RNA folding problem is significant because local base pairing and stacking interactions in single stranded RNA enable it to fold into many alternative conformations. Protein-mediated RNA remodeling is particularly important for understanding the function of regulatory RNAs, yet the mechanisms by which proteins can bias the folding free energy landscape are largely a mystery. We propose investigations of the homo-undecameric (11-mer) ring-forming Bacillus trp RNA binding attenuation protein (TRAP), its interactions with its activator ligand, tryptophan (Trp), its regulatory target, the trp leader RNA, and inhibitor protein Anti-TRAP. TRAP serves as a sensor of intracellular tryptophan metabolites (Trp), which can bind its 11 identical sites, activating it for binding to specific RNA sequences in the 5' untranslated region of the trp operon. RNA binding by Trp-activated TRAP results in remodeling of RNA secondary structures implicated in regulating transcription via aborted transcripts (termination). Because of its homo-oligomeric structure and heteromeric interactions, TRAP is an exceptional model system for studying mechanisms of both homotropic and heterotropic allosteric regulation.
The aims are to (1) Determine the mechanisms of allosteric communication in the homo-oligomeric ligand binding protein TRAP, and (2) Elucidate the role of TRAP-dependent RNA folding in regulating transcription of the trp operon.
The aims will be pursued by a combination of structural, thermodynamic, kinetic and biochemical experiments, including NMR spectroscopy, calorimetry, native mass spectrometry, co-transcriptional chemical structure probing, and in vivo and in vitro biochemical assays.
Most cellular proteins assemble into symmetric structures with multiple components so that interaction between them allows for more precise control of their function. This ?allosteric? control is widespread throughout biology but remains enigmatic and the topic of intense interest. The proposed experiments will provide unparalleled insights into the mechanisms of allostery and modulation of RNA structure, and will therefore broadly impact our understanding of biology and human health.