McElroy Craig, Manfredo Amanda, Wendt Alice, Gollnick Paul, Foster Mark
Ohio State Biochemistry Program, Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
J Mol Biol. 2002 Oct 25;323(3):463-73. doi: 10.1016/s0022-2836(02)00940-3.
The tryptophan biosynthesis genes of several Bacilli are controlled through terminator/anti-terminator transcriptional attenuation. This process is regulated by tryptophan-dependent binding of the trp RNA-binding attenuation protein (TRAP) to the leader region of the trp operon mRNA, precluding formation of the antiterminator RNA hairpin, and allowing formation of the less stable terminator hairpin. Crystal structures are available of TRAP in complex with tryptophan and in ternary complex with tryptophan and RNA. However, no structure of TRAP in the absence of tryptophan is available; thus, the mechanism of allostery remains unclear. We have used transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments to study the mechanism of ligand-mediated allosteric regulation in the 90.6kDa 11-mer TRAP. By recording a series of two-dimensional 15N-edited TROSY NMR spectra of TRAP from the thermophile Bacillus stearothermophilus over an extended range of temperatures, we have found tryptophan binding to be temperature-dependent, in agreement with the previously observed temperature-dependent RNA binding. Triple-resonance TROSY-based NMR spectra recorded at 55 degrees C have allowed us to obtain backbone resonance assignments for uniformly 2H,13C,15N-labeled TRAP in the inactive form and in the active form (free and bound to tryptophan). On the basis of ligand-dependent differential line-broadening and chemical shift perturbations, coupled with the results of proteolytic sensitivity measurements, we infer that tryptophan-modulated protein flexibility (dynamics) plays a central role in TRAP function by altering its RNA-binding affinity. Furthermore, because the crystal structures show that the ligand is buried completely in the bound state, we speculate that such dynamic behavior may be important to enable rapid response to changes in intracellular tryptophan levels. Thus, we propose that allosteric control of TRAP is accomplished by ligand-altered protein dynamics.
几种芽孢杆菌的色氨酸生物合成基因是通过终止子/抗终止子转录衰减来控制的。这一过程由色氨酸依赖的色氨酸RNA结合衰减蛋白（TRAP）与色氨酸操纵子mRNA的前导区结合来调节，阻止抗终止子RNA发夹结构的形成，并允许形成稳定性较低的终止子发夹结构。已有TRAP与色氨酸结合的晶体结构以及TRAP与色氨酸和RNA形成三元复合物的晶体结构。然而，尚无无色氨酸时TRAP的结构；因此，变构机制仍不清楚。我们利用基于横向弛豫优化光谱（TROSY）的核磁共振实验来研究90.6 kDa的11聚体TRAP中配体介导的变构调节机制。通过记录嗜热脂肪芽孢杆菌TRAP在较宽温度范围内的一系列二维15N编辑TROSY核磁共振谱，我们发现色氨酸结合具有温度依赖性，这与之前观察到的温度依赖性RNA结合一致。在55℃记录的基于三重共振TROSY的核磁共振谱使我们能够获得无活性形式和活性形式（游离和结合色氨酸）的均匀2H、13C、15N标记TRAP的主链共振归属。基于配体依赖性的差异线宽展宽和化学位移扰动，结合蛋白水解敏感性测量结果，我们推断色氨酸调节的蛋白质灵活性（动力学）通过改变其RNA结合亲和力在TRAP功能中起核心作用。此外，由于晶体结构表明配体在结合状态下完全被掩埋，我们推测这种动态行为可能对快速响应细胞内色氨酸水平的变化很重要。因此，我们提出TRAP的变构控制是通过配体改变的蛋白质动力学来实现的。
