从内源性基因表达数据推断RNA转录物特征和蛋白质表达谱之间的一般因果关系仍然是生物学中尚未解决的主要问题。分析来自异源基因的基因表达对解决这一问题具有重要的作用,但严重偏向于研究5个转录物区域的作用和原核生物。这里,我们采用合成生物学驱动的方法,系统地区分转录本的不同区域对基因表达的影响,直至240个核苷酸进入ORF。这使我们能够发现以前未探索的转录本区域中特征之间的新因果效应,和自然状态下的基因表达。我们合理地设计,constructed,并分析了病毒HRSVgp04基因ORF的383个基因变体,在真核酿酒酵母转录本的关键位置有多个同义突变。我们的结果表明,5'UTR的一些沉默突变可以对蛋白质水平产生高达15倍变化的戏剧性影响,即使在ORF5'末端下游超过120个核苷酸的位置中的同义突变也可以调节蛋白质水平高达160%-300%。我们证明,蛋白质水平和折叠能量之间的相关性随着内源性基因中后者的选择水平的重要性而增加,加强了ORF不同部分的折叠强度选择与翻译调节有关的概念。我们测量的蛋白质丰度与平均相对密码子解码时间显着相关(相关性高达r=0.62(p=0.0013)),基于内源基因中的核糖体密度(Ribo-Seq),支持以下猜想:翻译延伸和对tRNA库的适应可以以因果/直接方式修饰蛋白质水平。本报告提供了对转录本进化的更好理解,基因表达调控的设计原则,并提出了在真核生物中工程化合成基因表达的简单规则。
Deducing generic causal relations between RNA transcript features and protein expression profiles from endogenous gene expression data remains a major unsolved problem in biology. The analysis of gene expression from heterologous genes contributes significantly to solving this problem, but has been heavily biased toward the study of the effect of 5\' transcript regions and to prokaryotes. Here, we employ a synthetic biology driven approach that systematically differentiates the effect of different regions of the transcript on gene expression up to 240 nucleotides into the ORF. This enabled us to discover new causal effects between features in previously unexplored regions of transcripts, and gene expression in natural regimes. We rationally designed, constructed, and analyzed 383 gene variants of the viral HRSVgp04 gene ORF, with multiple synonymous mutations at key positions along the transcript in the eukaryote S. cerevisiae. Our results show that a few silent mutations at the 5\'UTR can have a dramatic effect of up to 15 fold change on protein levels, and that even synonymous mutations in positions more than 120 nucleotides downstream from the ORF 5\'end can modulate protein levels up to 160%-300%. We demonstrate that the correlation between protein levels and folding energy increases with the significance of the level of selection of the latter in endogenous genes, reinforcing the notion that selection for folding strength in different parts of the ORF is related to translation regulation. Our measured protein abundance correlates notably(correlation up to r = 0.62 (p=0.0013)) with mean relative codon decoding times, based on ribosomal densities (Ribo-Seq) in endogenous genes, supporting the conjecture that translation elongation and adaptation to the tRNA pool can modify protein levels in a causal/direct manner. This report provides an improved understanding of transcript evolution, design principles of gene expression regulation, and suggests simple rules for engineering synthetic gene expression in eukaryotes.