Positively charged residues are the major determinants of ribosomal velocity.
Positively charged residues are the major determinants of ribosomal velocity.
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DOI：
10.1371/journal.pbio.1001508
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发表时间：
2013
期刊：
影响因子：
9.8
通讯作者：
Hurst LD
中科院分区：
文献类型：
--
作者：
Charneski CA;Hurst LD
新生肽中带正电荷的氨基酸残基，而不是长期认为的RNA水平特征，减缓了核糖体。对于理解疾病的机制和转基因的设计，理解核糖体速度的决定因素是很重要的，因为翻译速率的变化对蛋白质折叠、错误衰减和定位都很重要。尽管核糖体的占有率沿着甚至是单个转录物都有很大的变化，但决定核糖体占有率的因素尚不清楚。我们使用酵母核糖体足迹分析的数据来研究这个问题。虽然密码子的使用被经典地认为是一个主要的决定因素，但我们没有发现这方面的证据。相比之下，我们发现，带正电荷的氨基酸大大阻碍核糖体下游的地方，他们被编码，一致的建议，带正电荷的残基与带负电荷的核糖体出口隧道相互作用。这种减缓是独立的，并大于平均效果，由于mRNA折叠。带电荷的氨基酸的影响是加性的，与核糖体占用预测良好的线性拟合带正电荷的残基的密度。因此，我们预期，翻译的poly-A尾，编码带正电荷的赖氨酸，无论阅读框架，将作为一个陷阱的核糖体，与实验数据一致。核糖体并不是以沿着转录物的恒定速率合成蛋白质，并且翻译速度的变化可以对该蛋白质的表达产生连锁影响，甚至改变其折叠或亚细胞定位。长期以来，人们一直认为RNA水平的特征调节翻译速率，无论是通过需要相对罕见的tRNA的密码子的存在而引起的延迟，还是通过物理上阻碍核糖体进展的mRNA折叠区域。相反，我们发现，它不是RNA水平的功能，但在已经翻译的蛋白质中的正电荷，大多数阻碍核糖体，可能是通过与带负电荷的核糖体出口隧道相互作用。我们发现，正电荷解释了核糖体停滞最常见的转录本内的网站。我们还表明，如果不考虑蛋白质电荷，人们可能会被误导，怀疑非最佳密码子的作用。最后，我们观察到聚腺苷酸尾提供了一个带大量正电荷的末端，无论它在哪个框架中被翻译。丢失的终止密码子或移码将导致停滞的核糖体，这与实验数据一致。
Positively charged amino acid residues in the nascent peptide, not RNA-level features as long thought, slow ribosomes. Both for understanding mechanisms of disease and for the design of transgenes, it is important to understand the determinants of ribosome velocity, as changes in the rate of translation are important for protein folding, error attenuation, and localization. While there is great variation in ribosomal occupancy along even a single transcript, what determines a ribosome's occupancy is unclear. We examine this issue using data from a ribosomal footprinting assay in yeast. While codon usage is classically considered a major determinant, we find no evidence for this. By contrast, we find that positively charged amino acids greatly retard ribosomes downstream from where they are encoded, consistent with the suggestion that positively charged residues interact with the negatively charged ribosomal exit tunnel. Such slowing is independent of and greater than the average effect owing to mRNA folding. The effect of charged amino acids is additive, with ribosomal occupancy well-predicted by a linear fit to the density of positively charged residues. We thus expect that a translated poly-A tail, encoding for positively charged lysines regardless of the reading frame, would act as a sandtrap for the ribosome, consistent with experimental data. Ribosomes do not synthesize protein at a constant rate along transcripts, and changes in translation speed can have knock-on consequences for the expression of that protein, even altering its folding or subcellular localization. It has long been thought that RNA-level features modulate translation rates, whether by delays incurred through the presence of codons that require relatively rare tRNAs, or by regions of mRNA folding that physically impede ribosomal progression. We find on the contrary that it is not RNA-level features but positive charges in the already translated protein that most retard ribosomes, possibly by interacting with the negatively charged ribosomal exit tunnel. We show that positive charge explains the sites where ribosomes stall most commonly within transcripts. We also show why, if protein charge were not considered, one could be misled into suspecting a role for non-optimal codons. Finally, we observe that the poly-A tail provides a massively positively charged terminus no matter in which frame it is translated. A missed stop codon or frameshifting would then lead to a stalled ribosome, which is consistent with experimental data.
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