Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation
Unwinding single RNA molecules using helicases involved in eukaryotic translation initiation
复制标题
DOI：
10.1016/j.jmb.2006.06.016
复制
发表时间：
2006-08-11
影响因子：
5.6
通讯作者：
McCarthy, John E. G.
中科院分区：
文献类型：
--
作者：
Marsden, Steven;Nardelli, Maria;McCarthy, John E. G.
真核核糖体的小（40 S）亚基在翻译起始密码子上起始之前可能必须从mRNA分子上的其5 '帽募集点扫描超过2000个核苷酸（> 600 nm）。与活细胞中的许多其他过程一样，包括转录，编辑，mRNA剪接，pre-rRNA加工，RNA运输和RNA衰变，扫描由解旋酶活性促进。然而，扫描的分子机制，包括解旋酶的作用，缺乏精确的定量数据。在这里，我们描述了一种新的原子力显微镜（AFM）为基础的程序来检查的两个酵母解旋酶，eIF 4A和DED 1，以前涉及的遗传和生化研究的翻译起始的作用。我们的研究结果表明，eIF 4A，特别是在它的“辅因子”eIF 4 B的存在下，促进ATP依赖性解旋的局部二级结构在长RNA分子的张力负荷下，虽然只有在高蛋白质：RNA的比例。因此，eIF 4A可能通过空间解旋机制仅能分离有限数量的碱基对。相比之下，Ded 1在降低（在14 nN（-1）的AFM加载速率下高达50 pN）破坏RNA茎环所需的力方面更有效，因此显示出促进快速解旋的显著动力学能力。这些单分子实验表明，Ded 1可能作为天然mRNA底物上更有效的解旋因子。(c)2006爱思唯尔有限公司保留所有权利。
The small (40 S) subunit of the eukaryotic ribosome may have to scan more than 2000 nucleotides (> 600 nm) from its 5'cap recruiting point on an mRNA molecule before initiating on a translation start codon. As with many other processes in living cells, including transcription, editing, mRNA splicing, pre-rRNA processing, RNA transport and RNA decay, scanning is facilitated by helicase activity. However, precise quantitative data on the molecular mechanism of scanning, including the roles of helicases, are lacking. Here, we describe a novel atomic force microscopy (AFM)-based procedure to examine the roles of two yeast helicases, eIF4A and Ded1, previously implicated in translation initiation by genetic and biochemical studies. Our results show that eIF4A, especially in the presence of its "cofactor" eIF4B, promotes ATP-dependent unwinding of localised secondary structure in long RNA molecules under tensional loading, albeit only at high protein:RNA ratios. Thus eIF4A can act to separate only a limited number of base-pairs, possibly via a steric unwinding mechanism. In contrast, Ded1 is more effective in reducing (by up to 50 pN at an AFM loading rate of 14 nNs(-1)) the force necessary to disrupt an RNA stem-loop, and thus shows significant kinetic competence to facilitate fast unwinding. These single molecule experiments indicate that Ded1 is likely to act as the more potent unwinding factor on natural mRNA substrates. (c) 2006 Elsevier Ltd. All rights reserved.