核糖体是活细胞的蛋白质制造工厂,它们以细胞中核苷酸的遗传密码子进行蛋白质的生产,当然,信使RNA(mRNA)提供蛋白质翻译的遗传密码,核糖体缠绕在信使RNA分子,通过识别起始和终止信号进行蛋白质的生产。如果一个信号缺失,蛋白质的生产就不能完成,这样一来,核糖体的生产模式就会被阻塞。
直到现在,我们并不清楚核糖体是如何克服这种蛋白质生产过程中的阻塞的,在修复过程中,即反式翻译过程中,一种额外的核酸分子(tmRNA)可以将mRNA分子和转移RNA分子(tRNA)联合起来,在蛋白质产生的过程中,转移RNA分子可以将正确的氨基酸分子转移到mRNA上,在蛋白质产生过程中,如果信号终止,tmRNA分子能够偷偷窜进来,解除蛋白质合成的封锁。但是tmRNA分子是如何穿过核糖体将其信息运输到mRNA上的,目前研究者并不清楚。
目前,此过程可以用低温电子显微镜进行记录,这种方法可以记录单一大分子组分之间的空间和时间上相互作用,这都可以通过速冻核糖体在负192摄氏度的液态乙烷中完成,而且数以千计的蛋白颗粒二维投影可以被反投影成其三维结构。运用低温电子显微镜技术,研究者可以详细记录核糖体、tmRNA、特殊蛋白SmbP和延伸因子G之间的相互作用。
在mRNA通道中,tmRNA可以悄悄地提供确实的信号信息,该通道径直穿过核糖体的中间,在小核糖体亚基的头部和身体结构域之间。研究者通过进行结构分析展示了核糖体和tmRNA之间在修复过程中的相互协作和相互作用。相关研究刊登在了近日的国际杂志Nature上。(生物谷:T.Shen编译)
doi:10.1038/nature11006
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The complex of tmRNA–SmpB and EF-G on translocating ribosomes
David J. F. Ramrath, Hiroshi Yamamoto, Kristian Rother, Daniela Wittek, Markus Pech, Thorsten Mielke, Justus Loerke, Patrick Scheerer, Pavel Ivanov, Yoshika Teraoka, Olga Shpanchenko, Knud H. Nierhaus & Christian M. T. Spahn
Bacterial ribosomes stalled at the 3′ end of malfunctioning messenger RNAs can be rescued by transfer-messenger RNA (tmRNA)-mediated trans-translation1, 2. The SmpB protein forms a complex with the tmRNA, and the transfer-RNA-like domain (TLD) of the tmRNA then enters the A site of the ribosome. Subsequently, the TLD–SmpB module is translocated to the P site, a process that is facilitated by the elongation factor EF-G, and translation is switched to the mRNA-like domain (MLD) of the tmRNA. Accurate loading of the MLD into the mRNA path is an unusual initiation mechanism. Despite various snapshots of different ribosome–tmRNA complexes at low to intermediate resolution3, 4, 5, 6, 7, it is unclear how the large, highly structured tmRNA is translocated and how the MLD is loaded. Here we present a cryo-electron microscopy reconstruction of a fusidic-acid-stalled ribosomal 70S–tmRNA–SmpB–EF-G complex (carrying both of the large ligands, that is, EF-G and tmRNA) at 8.3 Å resolution. This post-translocational intermediate (TIPOST) presents the TLD–SmpB module in an intrasubunit ap/P hybrid site and a tRNAfMet in an intrasubunit pe/E hybrid site. Conformational changes in the ribosome and tmRNA occur in the intersubunit space and on the solvent side. The key underlying event is a unique extra-large swivel movement of the 30S head, which is crucial for both tmRNA–SmpB translocation and MLD loading, thereby coupling translocation to MLD loading. This mechanism exemplifies the versatile, dynamic nature of the ribosome, and it shows that the conformational modes of the ribosome that normally drive canonical translation can also be used in a modified form to facilitate more complex tasks in specialized non-canonical pathways.