California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA.
DGIMI, Univ. Montpellier, INRA, Montpellier, France.
Nat Biotechnol. 2018 Nov;36(10):1005-1015. doi: 10.1038/nbt.4238. Epub 2018 Sep 24.
Comparative analyses of natural and mutated sequences have been used to probe mechanisms of gene expression, but small sample sizes may produce biased outcomes. We applied an unbiased design-of-experiments approach to disentangle factors suspected to affect translation efficiency in E. coli. We precisely designed 244,000 DNA sequences implementing 56 replicates of a full factorial design to evaluate nucleotide, secondary structure, codon and amino acid properties in combination. For each sequence, we measured reporter transcript abundance and decay, polysome profiles, protein production and growth rates. Associations between designed sequences properties and these consequent phenotypes were dominated by secondary structures and their interactions within transcripts. We confirmed that transcript structure generally limits translation initiation and demonstrated its physiological cost using an epigenetic assay. Codon composition has a sizable impact on translatability, but only in comparatively rare elongation-limited transcripts. We propose a set of design principles to improve translation efficiency that would benefit from more accurate prediction of secondary structures in vivo.
对天然和突变序列的比较分析被用于探究基因表达的机制，但小样本量可能会产生有偏的结果。我们应用了一种无偏的实验设计方法来解析被怀疑影响大肠杆菌翻译效率的因素。我们精确设计了 244000 个 DNA 序列，实施了一个全因子设计的 56 个重复实验，以评估核苷酸、二级结构、密码子和氨基酸性质的综合影响。对于每个序列，我们测量了报告转录物的丰度和衰减、多核糖体谱、蛋白质产量和生长速率。设计序列性质与这些后续表型之间的关联主要由转录物中的二级结构及其相互作用决定。我们证实了转录物结构通常限制翻译起始，并使用表观遗传测定法证明了其生理代价。密码子组成对可翻译性有相当大的影响，但仅在相对罕见的延伸受限转录物中。我们提出了一组设计原则来提高翻译效率，这将受益于更准确地预测体内的二级结构。
