Holschuh K, Gassen H G
J Biol Chem. 1982 Feb 25;257(4):1987-92.
The translocation of the mRNA in relation to the ribosome during peptide synthesis represents an example for a mechanochemical reaction in which the chemical bond energy of GTP is transformed into coordinated motion. We demonstrate here that translocation can be explained simply by binding equilibria between the tRNA, the mRNA, and their binding sites on the ribosome. The presence of two cognate tRNAs shifts the association constant for the 70 S ribosome . AUGU3 complex from 6.8 x 10(5) to 2.2 x 10(8) M-1. The elongation factor G and GTP or guanosine-5'-(beta,gamma-methylene)triphosphate GMP-PCP) displace the methionine tRNAs which can be formylated (tRNAfMet) from the quaternary complex 70 S . AUGU3 . tRNAfMet . tRNAPhe. Only the ternary complex Phe-tRNAPhe . elongation factor Tu . GMP-PCP shows an absolute preference for the aminoacyl-tRNA binding site (A site) (K a = 6.6 x 10(6) M-1). AcPhe-tRNAPhe, (N alpha-acetylphenylalanyl-tRNA) an analogue of a peptidyl-tRNA exhibits a 20-fold higher affinity to the peptidyl-tRNA binding site (P site) (K a = 3.5 x 10(6) M-1) as against the A site (K a = 1.8 x 10(6) M-1) at 8 mM Mg2+. Compared to aminoacyl-tRNA and tRNA, peptidyl-tRNA shows a 3- to 15-fold higher affinity toward complementary oligonucleotides both in the binary complex and in the presence of 70 S ribosomes (UUCA . AcPhe-tRNAPhe: K a = 1.9 x 10(5) M-1), UUCA . tRNAPhe:K a = 3.2 x 10(4) M-1). This indicates a stabilization of the peptidyl-tRNA . mRNA complex during translocation. Our data support a concept of mRNA translocation in which the removal of the deacylated tRNA from the P site requires GTP energy and a peptidyl-tRNA . mRNA complex diffuses from its low affinity site (A) to its high affinity binding site (P).
在肽合成过程中，mRNA相对于核糖体的易位代表了一种机械化学反应的例子，其中GTP的化学键能转化为协同运动。我们在此证明，易位可以简单地通过tRNA、mRNA及其在核糖体上的结合位点之间的结合平衡来解释。两个同源tRNA的存在使70S核糖体.AUGU3复合物的缔合常数从6.8×10⁵ M⁻¹变为2.2×10⁸ M⁻¹。延伸因子G和GTP或鸟苷-5'-（β，γ-亚甲基）三磷酸（GMP-PCP）将可被甲酰化的甲硫氨酸tRNA（tRNAfMet）从四元复合物70S.AUGU3.tRNAfMet.tRNAPhe中置换出来。只有三元复合物苯丙氨酰-tRNAPhe.延伸因子Tu.GMP-PCP对氨酰-tRNA结合位点（A位点）表现出绝对偏好（Ka = 6.6×10⁶ M⁻¹）。乙酰苯丙氨酰-tRNAPhe（Nα-乙酰苯丙氨酰-tRNA），一种肽基-tRNA的类似物，在8 mM Mg²⁺条件下，对肽基-tRNA结合位点（P位点）的亲和力比对A位点的亲和力高20倍（Ka = 3.5×10⁶ M⁻¹对Ka = 1.8×10⁶ M⁻¹）。与氨酰-tRNA和tRNA相比，肽基-tRNA在二元复合物以及存在70S核糖体的情况下，对互补寡核苷酸的亲和力高3至15倍（UUCA.乙酰苯丙氨酰-tRNAPhe：Ka = 1.9×10⁵ M⁻¹），UUCA.tRNAPhe：Ka = 3.2×10⁴ M⁻¹）。这表明在易位过程中肽基-tRNA.mRNA复合物得到了稳定。我们的数据支持一种mRNA易位的概念，即从P位点去除脱酰基tRNA需要GTP能量，并且肽基-tRNA.mRNA复合物从其低亲和力位点（A）扩散到其高亲和力结合位点（P）。
