Real-time tRNA transit on single translating ribosomes at codon resolution.
Real-time tRNA transit on single translating ribosomes at codon resolution.
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作者：
核糖体的翻译通过一种复杂的机制发生，涉及多个核酸和蛋白质配体的协调相互作用。在这里，我们使用零模波导(ZMW)和复杂的检测仪器来实时观察生理相关(μM)配体浓度下的平移。每个密码子的翻译通过tRNA-标记有不同荧光团的tRNA-与翻译核糖体的稳定结合来监测，从而允许直接检测与核糖体结合的tRNA分子的身份，从而检测潜在的mRNA序列。我们观察了tRNAs在单个翻译核糖体上的转运，并确定了在mRNA的每个密码子上同时与核糖体结合的tRNA分子的数量。我们的结果表明，核糖体只被两个tRNA短暂占据，脱酰化的tRNA从E位释放出来是与A位tRNA结合解偶联的，并在易位后迅速发生。这些方法在研究信使核糖核酸序列，以及翻译的机制和调控方面有着广泛的应用。
Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we have used zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically-relevant (μM) ligand concentrations. Translation at each codon is monitored by stable binding of tRNAs – labeled with distinct fluorophores – to translating ribosomes, allowing direct detection of the identity of tRNA molecules bound to the ribosome, and therefore, the underlying mRNA sequence. We observe the transit of tRNAs on single translating ribosomes and have determined the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA. Our results show that ribosomes are only briefly occupied by two tRNAs and that release of deacylated tRNA from the E site is uncoupled from binding of A-site tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.
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影响因子：
3.4
作者：
Chan, V;Graves, DJ;McKenzie, SE
通讯作者：
McKenzie, SE
DOI：
10.1073/pnas.0710982105
发表时间：
2008-01-29
影响因子：
11.1
作者：
Korlach, Jonas;Marks, Patrick J.;Turner, Stephen W.
通讯作者：
Turner, Stephen W.
影响因子：
3.4
作者：
Aitken, Colin Echeverria;Marshall, R. Andrew;Puglisi, Joseph D.
通讯作者：
Puglisi, Joseph D.
影响因子：
4.5
作者：
Sanders, Christina L.;Curran, James F.
通讯作者：
Curran, James F.
影响因子：
16
作者：
Marshall, R. Andrew;Aitken, Colin Echeverria;Puglisi, Joseph D.
通讯作者：
Puglisi, Joseph D.