Holschuh K, Bonin J, Gassen H G
Biochemistry. 1980 Dec 9;19(25):5857-64. doi: 10.1021/bi00566a030.
We try to mimic the unidirectional sliding-type movement of the PP-tRNA . mRNA complex with respect to the ribosome by looking at the effect of different combinations of cognate tRNAs on the stability of the 70S-AUGUn complex. The association constant for the binary complex 70S-AUGU3 was determined as 6.8 x 10(5) M-1. Addition of tRNAfMet resulted in a 67-fold increase in the association constant, which with both cognate tRNAs is revised to Kassoc = 2.2 x 10(8) M-1. Increasing the chain length of the oligonucleotide from AUGU3 to AUGU13 did not further raise the association constant. The data indicate that the stability of the 70S ribosome . mRNA interaction is governed by the presence of the cognate tRNAs and is topographically restricted to the decoding domains. Since a peptidyl group in the tRNA increases the affinity of AUGU3 for the ribosome by up to 15-fold, we conclude that the affinity of the peptidyl transfer center for the peptidyl moiety pulls the PP-tRNA . mRNA complex from the A (aminoacyl-tRNA) site to the P (peptidyl-tRNA) site. EF-G . GTP or EF-G . GMPPCP 5'-(beta, gamma-methylene)triphosphate] displace tRNAfMet from the quaternary complex 70S . AUGUn . tRNAfMet . tRNAPhe (n = 3 and 6) at Mg2+ less than 25 mM. From the amount of EF-G . GTP bound to a 70S ribosome, it follows that the elongation factor replaces the deacylated tRNA in a stoichiometric way. These data indicate that the EF-G . GTP-dependent release of the deacylated tRNA from the P site, followed by removal of EF-G . GDP from the 50S subunit, is sufficient to trigger the translocation of the mRNA . PP-tRNA complex.
我们通过观察同源tRNA的不同组合对70S - AUGUn复合物稳定性的影响，来尝试模拟PP - tRNA·mRNA复合物相对于核糖体的单向滑动式运动。二元复合物70S - AUGU3的结合常数测定为6.8×10⁵ M⁻¹。添加起始甲硫氨酸tRNA（tRNAfMet）导致结合常数增加了67倍，与两种同源tRNA结合时，结合常数修正为Kassoc = 2.2×10⁸ M⁻¹。将寡核苷酸链长度从AUGU3增加到AUGU13并没有进一步提高结合常数。数据表明，70S核糖体·mRNA相互作用的稳定性受同源tRNA的存在控制，并且在拓扑结构上局限于解码结构域。由于tRNA中的肽基使AUGU3对核糖体的亲和力增加了多达15倍，我们得出结论，肽基转移中心对肽基部分的亲和力将PP - tRNA·mRNA复合物从A（氨酰 - tRNA）位点拉到P（肽酰 - tRNA）位点。在镁离子浓度低于25 mM时，延伸因子G（EF - G）·鸟苷三磷酸（GTP）或EF - G·5'-（β，γ - 亚甲基）三磷酸鸟苷（GMPPCP）会从四元复合物70S·AUGUn·tRNAfMet·苯丙氨酸tRNA（tRNAPhe，n = 3和6）中取代tRNAfMet。根据结合到70S核糖体上的EF - G·GTP的量，可以得出延伸因子以化学计量的方式取代脱酰基tRNA。这些数据表明，EF - G·GTP依赖性地将脱酰基tRNA从P位点释放，随后从50S亚基上去除EF - G·鸟苷二磷酸（GDP）足以触发mRNA·PP - tRNA复合物的易位。
