Rheinberger H J, Nierhaus K H
Max-Planck-Institut für Molekulare Genetik, Abt. Wittmann, Berlin-Dahlem, West Germany.
J Biomol Struct Dyn. 1987 Oct;5(2):435-46. doi: 10.1080/07391102.1987.10506403.
Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM. The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM. At 4 mM the inactivation amounts to about 80%. The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site. The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM. The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4. In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J. Biol. Chem. 261, 9133 (1986] are demonstrated. 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation. 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites). The quality of an in vitro system for protein synthesis can be assessed by two criteria. First, the incubation conditions must allow a near quantitative tRNA binding. Secondly, protein synthesis should proceed with near in vivo rate and accuracy. The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.
在标准条件下（Mg2+/150 mM NH4+），核糖体能够在12至15 mM的Mg2+浓度下定量参与tRNA结合。当Mg2+浓度降至10 mM以下时，总的聚尿苷酸（poly(U)）指导的苯丙氨酸掺入以及tRNA与P、E或A位点的结合程度会以平行方式降低。在4 mM时，失活程度约为80%。所有三个结合位点的协同失活伴随着将A位点结合的乙酰苯丙氨酰 - tRNA（AcPhe - tRNA）转运至P位点能力的日益受损。转运效率在10 mM Mg2+时就已降低，并在6 - 8 mM时完全受阻。当测定体系中存在多胺精胺（0.6 mM）和亚精胺（0.4 mM）时，在6 mM Mg2+时观察到的严重失活现象消失；tRNA结合再次变为定量，总的苯丙氨酸合成甚至比不存在多胺时高出4倍。在多胺和低Mg2+（3和6 mM）存在的情况下，变构三位点模型的两个基本特征（莱茵伯格和尼尔豪斯，《生物化学杂志》261, 9133 (1986)）得以证明。1）脱酰基tRNA不会从P位点释放，而是在转运过程中转移至E位点。2）E位点的占据会降低A位点的亲和力，反之亦然（E位点和A位点之间的变构相互作用）。蛋白质合成体外系统的质量可以通过两个标准来评估。首先，孵育条件必须允许近乎定量的tRNA结合。其次，蛋白质合成应以接近体内的速率和准确性进行。3 mM Mg2+/NH4+/多胺系统似乎是目前在这两个要求之间的最佳折衷方案。
