de Smit Maarten H, van Duin Jan
Genexpress, Gorlaeus Laboratories, Leiden Institute of Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands.
J Mol Biol. 2003 Aug 22;331(4):737-43. doi: 10.1016/s0022-2836(03)00809-x.
We have previously shown that stable base-pairing at a translational initiation site in Escherichia coli can inhibit translation by competing with the binding of ribosomes. When the base-pairing is not too strong, this competition is won by the ribosomes, resulting in efficient translation from a structured ribosome binding site (RBS). We now re-examine these results in the light of RNA folding kinetics and find that the window during which a folded RBS is open is generally much too short to recruit a 30S ribosomal subunit from the cytoplasm. We argue that to achieve efficient expression, a 30S subunit must already be in contact with the mRNA while this is still folded, to shift into place as soon as the structure opens. Single-stranded regions flanking the structure may constitute a standby site, to which the 30S subunit can attach non-specifically. We propose a steady-state kinetic model for the early steps of translational initiation and use this to examine various quantitative aspects of standby binding. The kinetic model provides an explanation of why the earlier equilibrium competition model predicted implausibly high 30S-mRNA affinities. Because all RNA is structured to some degree, standby binding is probably a general feature of translational initiation.
我们之前已经表明，大肠杆菌中翻译起始位点处稳定的碱基配对可通过与核糖体结合竞争来抑制翻译。当碱基配对不太强时，核糖体赢得这种竞争，从而从结构化的核糖体结合位点（RBS）进行高效翻译。我们现在根据RNA折叠动力学重新审视这些结果，发现折叠后的RBS开放的时间窗口通常太短，无法从细胞质中招募30S核糖体亚基。我们认为，为了实现高效表达，30S亚基必须在mRNA仍折叠时就已经与之接触，以便在结构打开时立即就位。结构两侧的单链区域可能构成一个备用位点，30S亚基可以非特异性地附着于此。我们提出了一个翻译起始早期步骤的稳态动力学模型，并用它来研究备用结合的各种定量方面。该动力学模型解释了为什么早期的平衡竞争模型预测出了高得难以置信的30S - mRNA亲和力。由于所有RNA在某种程度上都是结构化的，备用结合可能是翻译起始的一个普遍特征。
