Studies of anti-TRAP (AT) protein and its interactions with TRAP.

摘要

In Bacillus subtilis, genes involved in tryptophan biosynthesis and transport (trp) are regulated in response to changes in the intracellular levels of free L-tryptophan and to accumulation of uncharged tRNATrp. Two regulatory proteins, TRAP (trp RNA-binding attenuation protein) and Anti-TRAP (AT), modulate expression of trp genes according to these changes. TRAP down-regulate transcription and translation of trp genes in response to elevated levels of tryptophan. Upon binding to tryptophan, TRAP is activated to bind several mRNAs, which wrap around the outer periphery of the protein. AT binds to tryptophan-activated TRAP and inhibits it from binding to the RNA targets. Although how TRAP binds to and controls trp gene expression have been well understood, relatively little is known about the mechanism by which AT interacts with TRAP and prevents TRAP binding to RNA.; In this dissertation we studied the interactions between AT and TRAP. We examined the thermodynamics and kinetics of the AT-TRAP interaction, and determined the dependence of the interaction on tryptophan concentration, ionic strength, pH, and temperature. The results reveal that electrostatic interactions are not essential in stabilizing the AT-TRAP complex. Rather, AT binding to TRAP involves hydrophobic interactions between the two proteins. Such interactions are entropically driven, possibly resulted from release of ordered water molecules upon complex formation. Analysis of the binding data using different kinetic models indicates that AT binds to TRAP as trimers (AT3) and dodecamers (AT12).; The residues of AT that are involved in binding to TRAP have not been identified. Thus we used alanine mutagenesis to identify these residues. Our results demonstrate that several residues at the top area of the AT3 cone structure are needed for interaction with TRAP. We then used fluorescence labeling to further investigate the role of the top area of the AT3 in binding to TRAP. We found that residues at the top region are near TRAP in the AT-TRAP complex. In addition, in vivo studies of the activity of several alanine mutant AT proteins confirmed the importance of the top residues. Together, our results suggest that residues at the top area of AT3 structure are directly involved in interaction with TRAP. Studies in this dissertation provide information for understanding the AT-TRAP interaction.