Studer Sean M, Joseph Simpson
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
Mol Cell. 2006 Apr 7;22(1):105-15. doi: 10.1016/j.molcel.2006.02.014.
Translation initiation is a key step for regulating the level of numerous proteins within the cell. In bacteria, the 30S initiation complex directly binds to the translation initiation region (TIR) of the mRNA. How the ribosomal 30S subunit assembles on highly structured TIR is not known. Using fluorescence-based experiments, we assayed 12 different mRNAs that form secondary structures with various stabilities and contain Shine-Dalgarno (SD) sequences of different strengths. A strong correlation was observed between the stability of the mRNA structure and the association and dissociation rate constants. Interestingly, in the presence of initiation factors and initiator tRNA, the association kinetics of structured mRNAs showed two distinct phases. The second phase was found to be important for unfolding structured mRNAs to form a stable 30S initiation complex. We show that unfolding of structured mRNAs requires a SD sequence, the start codon, fMet-tRNA(fMet), and the GTP bound form of initiation factor 2 bound to the 30S subunit.
翻译起始是调控细胞内众多蛋白质水平的关键步骤。在细菌中，30S起始复合物直接结合到mRNA的翻译起始区域（TIR）。核糖体30S亚基如何在高度结构化的TIR上组装尚不清楚。我们使用基于荧光的实验，检测了12种不同的mRNA，它们形成具有不同稳定性的二级结构，并包含不同强度的Shine-Dalgarno（SD）序列。在mRNA结构的稳定性与缔合和解离速率常数之间观察到了很强的相关性。有趣的是，在起始因子和起始tRNA存在的情况下，结构化mRNA的缔合动力学表现出两个不同的阶段。发现第二阶段对于解开结构化mRNA以形成稳定的30S起始复合物很重要。我们表明，结构化mRNA的解折叠需要一个SD序列、起始密码子、fMet-tRNA(fMet)以及与30S亚基结合的GTP结合形式的起始因子2。
