Southworth Daniel R, Brunelle Julie L, Green Rachel
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, MD 21205, USA.
J Mol Biol. 2002 Dec 6;324(4):611-23. doi: 10.1016/s0022-2836(02)01196-8.
Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.
在没有延伸因子EFG和EFTu（以及来自GTP水解的能量）的情况下，核糖体可利用多聚尿苷模板翻译出多聚苯丙氨酸，而用对氯汞苯甲酸（pCMB）等硫醇特异性试剂修饰核糖体可促进这一过程。在此，我们研究了修饰核糖体的翻译循环，结果表明肽键形成和tRNA结合基本不受影响，而mRNA:tRNA复合物的易位则因pCMB修饰而显著促进。我们观察到的易位运动通过多种标准得以验证，包括翻译的持续性、沿特定mRNA模板移动（三个核苷酸）的准确性以及对抗生素的敏感性。对修饰核糖体的表征显示，核糖体的蛋白质含量并未减少，但其亚基结合特性却严重受损。这些数据表明，核糖体界面区域的分子靶点（核糖体蛋白）是影响mRNA:tRNA复合物易位的关键障碍。
