Jagath J R, Rodnina M V, Lentzen G, Wintermeyer W
Institute of Molecular Biology, University of Witten/Herdecke, Germany.
Biochemistry. 1998 Nov 3;37(44):15408-13. doi: 10.1021/bi981523a.
The bacterial signal recognition particle (SRP) is an RNA-protein complex. In Escherichia coli, the particle consists of a 114 nt RNA, a 4.5S RNA, and a 48 kDa GTP-binding protein, Ffh. GDP-GTP exchange on, and GTP hydrolysis by, Ffh are thought to regulate SRP function in membrane targeting of translating ribosomes. In the present paper, we report the equilibrium and kinetic constants of guanine nucleotide binding to Ffh in different functional complexes. The association and dissociation rate constants of GTP/GDP binding to Ffh were measured using a fluorescent analogue of GTP/GDP, mant-GTP/GDP. For both nucleotides, association and dissociation rate constants were about 10(6) M-1 s-1 and 10 s-1, respectively. The equilibrium constants of nonmodified GTP and GDP binding to Ffh alone and in SRP, and in the complex with the ribosomes were measured by competition with mant-GDP. In all cases, the same 1-2 microM affinity for GTP and GDP was observed. Binding of both GTP and GDP to Ffh was independent of Mg2+ ions. The data suggest that, at conditions in vivo, (i) there will be rapid spontaneous GDP-GTP exchange, and (ii) the GTP-bound form of Ffh, or of SRP, will be predominant.
细菌信号识别颗粒（SRP）是一种RNA-蛋白质复合物。在大肠杆菌中，该颗粒由一个114个核苷酸的RNA、一个4.5S RNA和一个48 kDa的GTP结合蛋白Ffh组成。Ffh上的GDP-GTP交换以及Ffh催化的GTP水解被认为在翻译核糖体的膜靶向中调节SRP功能。在本文中，我们报道了鸟嘌呤核苷酸在不同功能复合物中与Ffh结合的平衡常数和动力学常数。使用GTP/GDP的荧光类似物mant-GTP/GDP测量了GTP/GDP与Ffh结合和解离的速率常数。对于这两种核苷酸，结合和解离速率常数分别约为10⁶ M⁻¹ s⁻¹和10 s⁻¹。通过与mant-GDP竞争，测量了未修饰的GTP和GDP单独与Ffh结合、在SRP中以及与核糖体复合物中结合的平衡常数。在所有情况下，观察到对GTP和GDP具有相同的1 - 2 μM亲和力。GTP和GDP与Ffh的结合均不依赖于Mg²⁺离子。数据表明，在体内条件下，（i）会有快速的自发GDP-GTP交换，并且（ii）Ffh或SRP的GTP结合形式将占主导。
