Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.
Distinct stages of the translation elongation cycle revealed by sequencing ribosome-protected mRNA fragments.
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DOI：
10.7554/elife.01257
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发表时间：
2014-05-09
期刊：
影响因子：
7.7
通讯作者：
Brown PO
中科院分区：
文献类型：
--
作者：
Lareau LF;Hite DH;Hogan GJ;Brown PO
在翻译延伸期间，核糖体沿着其mRNA模板棘轮移动，结合每个新的氨基酸并从一个密码子转移到下一个密码子。延伸周期需要核糖体剧烈的结构重排。我们在这里展示了对受核糖体保护的mRNA片段的深度测序，不仅揭示了每个核糖体的位置，而且出乎意料地揭示了其延伸周期的特定阶段。测序揭示了两个不同的核糖体足迹群，28-30核苷酸和20-22核苷酸长，代表了不同状态的翻译核糖体，被特定的延伸抑制剂不同地稳定。我们发现小足迹和大足迹的平衡因密码子而异，并与翻译速度相关。在单密码子分辨率下可视化核糖体在延伸过程中的构象变化的能力，为研究翻译的详细动力学提供了一种新的方法，并为识别影响延伸周期中每个步骤的因素提供了一种新的探针。要从基因中制造蛋白质，首先要将基因转录成信使RNA （mRNA）分子，然后通过称为核糖体的分子机器。核糖体每次以三个字母为一组读取信使rna中的遗传密码，每个三个字母（或密码子）代表一个氨基酸。然后核糖体将相关的氨基酸连接在一起形成蛋白质。核糖体平均每秒处理6个氨基酸，但信使rna并不是以恒定的速率通过的。相反，核糖体改变它的形状，沿着mRNA从一个密码子到下一个密码子：然后它读取新的密码子，并在蛋白质上添加另一个氨基酸。然而，这个棘轮过程的许多细节尚不完全清楚。在这项研究中，Lareau， Hite等人使用了一种称为“核糖体分析”的技术来探索核糖体沿着mRNA分子的运动。首先，所有在核糖体中没有受到保护的mRNA分子片段都被化学破坏了。然后读取受保护片段的序列并与全长基因序列进行匹配。受保护的碎片有两种不同的大小：一些大约有28-30个字母长，另一些大约有20-22个字母长。Lareau， Hite等人认为，这些不同片段大小的发生是因为核糖体在沿着mRNA棘轮移动时，在每个密码子上在两种形状之间切换，因此它保护不同长度的mRNA。在之前的核糖体分析实验中，这些片段都大约有28个字母长；但这些实验在测量片段的长度之前，使用了一种化学物质来阻止核糖体沿着mrna的进程。Lareau， Hite等人认为，这种化学物质在使核糖体停止时将核糖体锁定在相同的形状，因此受保护的片段总是具有相同的长度。此外，其他阻止核糖体的化学物质似乎将这个分子机器锁定在另一种形状，因此它只能保护较短的片段。Lareau、Hite等人的发现表明，核糖体分析实验可以揭示的不仅仅是核糖体在mRNA分子上的位置。对核糖体棘轮过程不同阶段的进一步研究将有助于揭示核糖体将mRNA翻译成蛋白质的速度如何在mRNA序列本身中编码。DOI: http://dx.doi.org/10.7554/eLife.01257.002
During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28–30 nucleotides and 20–22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. DOI: http://dx.doi.org/10.7554/eLife.01257.001 To make a protein from a gene, the gene is first transcribed to produce a molecule of messenger RNA (mRNA), which then passes through a molecular machine called a ribosome. The ribosome reads the genetic code in the mRNA in groups of three letters at a time, and each triplet of letters (or codon) represents an amino acid. The ribosome then joins the relevant amino acids together to build a protein. The ribosome processes about six amino acids per second, on average, but the mRNA is not fed through at a constant rate. Instead, the ribosome changes its shape to ratchet along the mRNA from one codon to the next: it then reads the new codon and adds another amino acid to the protein. However, many of the details of this ratcheting process are not fully understood. In this study, Lareau, Hite et al. have used a technique called ‘ribosome profiling’ to explore the movement of ribosomes along mRNA molecules. First, all of the pieces of mRNA molecules that are not protected inside a ribosome were chemically destroyed. The sequences of the protected fragments were then read and matched to the full-length gene sequences. The protected fragments came in two different sizes: some were about 28–30 letters long, and others were about 20–22 letters long. Lareau, Hite et al. suggest that these different fragment sizes occur because the ribosome switches between two shapes at each codon as it ratchets along the mRNA, and so it protects different lengths of mRNA. In previous ribosome-profiling experiments, the fragments had all been about 28 letters long; but these experiments had used a chemical to halt the progress of the ribosomes along the mRNAs before measuring the length of the fragments. Lareau, Hite et al. argue that this chemical locks the ribosome in the same shape when it brings the ribosome to a halt, and so the protected fragments always have the same length. Further, other chemicals that halt ribosomes appear to lock this molecular machine in the other shape, and so it can only protect the shorter fragments. The findings of Lareau, Hite et al. show that ribosomal profiling experiments can reveal much more than simply where a ribosome is on an mRNA molecule. Further study into the different stages of the ribosome ratcheting process will help uncover how the speed that a ribosome translates an mRNA into a protein can be encoded in the mRNA sequence itself. DOI: http://dx.doi.org/10.7554/eLife.01257.002