Arava Yoav, Wang Yulei, Storey John D, Liu Chih Long, Brown Patrick O, Herschlag Daniel
Department of Biochemistry, Stanford University, Stanford, CA 94305-5307, USA.
Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3889-94. doi: 10.1073/pnas.0635171100. Epub 2003 Mar 26.
We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.
我们分析了快速生长的酿酒酵母中每个mRNA的翻译状态。通过在蔗糖梯度上进行速度沉降分离mRNA，并通过定量微阵列分析对梯度中的14个组分进行分析，从而获得数千个基因的核糖体与mRNA结合情况的图谱。对于大多数基因而言，大多数mRNA分子都与核糖体结合，并且可能正在进行翻译。这种系统的方法使我们能够识别出具有异常行为的基因。对于43个基因，大多数mRNA分子不与核糖体结合，这表明它们可能受到翻译调控。对于53个基因，包括已知翻译调控在调控中起关键作用的三个基因GCN4、CPA1和ICY2，大多数mRNA分子与单个核糖体结合。与mRNA结合的核糖体数量随着假定蛋白质编码序列长度的增加而增加，这与翻译较长编码序列的核糖体具有更长的转运时间一致。几乎所有mRNA上核糖体分布的密度（即每单位开放阅读框长度的核糖体数量）远低于最大堆积密度，这与起始作为翻译的限速步骤一致。全局分析揭示了一个意外的相关性：核糖体密度随着开放阅读框长度的增加而降低。文中讨论了解释这一惊人观察结果的模型。
