Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae.
Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae.
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Kozak 序列上游的核苷酸强烈影响酿酒酵母中的基因表达。
DOI：
10.1186/s13036-017-0068-1
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发表时间：
2017
影响因子：
5.6
通讯作者：
Marchisio MA
中科院分区：
文献类型：
--
作者：
Li J;Liang Q;Song W;Marchisio MA
在酿酒酵母中，如同在所有真核生物中一样，5‘-非翻译区对翻译起始起着重要的作用。然而，决定核糖体与mRNA结合的效率、核糖体通过5‘-UTR延长以及稳定的翻译起始复合体形成的模式和机制尚不清楚。在酿酒酵母中高表达的基因似乎偏爱富有腺嘌呤而缺乏鸟嘌呤的5‘-UTR，特别是在Kozak序列中，它大约占据起始密码子上游的前六个核苷酸。我们测量了58个合成版本的酿酒酵母最小Cyc1启动子(PCyC1min)产生的荧光，每个版本包含不同的5‘-UTR。首先，我们用腺嘌呤取代了最初的pCyC1min5‘-UTR的最后15个核苷酸--这是一个理论上高基因表达的最佳配置。接下来，我们对它进行了单点和多点突变。蛋白质合成受到Kozak序列上游单点和多点突变的高度影响。RNA折叠模拟显示，在−15和−9位置之间，三个以上的腺嘌呤突变为鸟嘌呤，从而导致基因二级结构的显著变化。此外，点突变的影响被证明是强烈的上下文依赖性，表明仅仅位于起始密码子上游的腺嘌呤本身并不能保证基因表达的增加，正如先前所说的那样。通过作用于Kozak序列上游的核苷酸，可以构建不同翻译起始率的新的合成真核生物启动子。翻译效率可能会受到起始密码子上游5‘-UTR的另一部分的影响。深入了解5‘-非编码区在基因表达中的作用将改进酵母合成基因回路中启动子的选择和使用标准。本文的在线版本(doi：10.1186/s13036-0170068-1)包含补充材料，授权用户可以使用。
In the yeast Saccharomyces cerevisiae, as in every eukaryotic organism, the mRNA 5′-untranslated region (UTR) is important for translation initiation. However, the patterns and mechanisms that determine the efficiency with which ribozomes bind mRNA, the elongation of ribosomes through the 5′-UTR, and the formation of a stable translation initiation complex are not clear. Genes that are highly expressed in S. cerevisiae seem to prefer a 5′-UTR rich in adenine and poor in guanine, particularly in the Kozak sequence, which occupies roughly the first six nucleotides upstream of the START codon. We measured the fluorescence produced by 58 synthetic versions of the S. cerevisiae minimal CYC1 promoter (pCYC1min), each containing a different 5′-UTR. First, we replaced with adenine the last 15 nucleotides of the original pCYC1min 5′-UTR—a theoretically optimal configuration for high gene expression. Next, we carried out single and multiple point mutations on it. Protein synthesis was highly affected by both single and multiple point mutations upstream of the Kozak sequence. RNAfold simulations revealed that significant changes in the mRNA secondary structures occur by mutating more than three adenines into guanines between positions −15 and −9. Furthermore, the effect of point mutations turned out to be strongly context-dependent, indicating that adenines placed just upstream of the START codon do not per se guarantee an increase in gene expression, as previously suggested. New synthetic eukaryotic promoters, which differ for their translation initiation rate, can be built by acting on the nucleotides upstream of the Kozak sequence. Translation efficiency could, potentially, be influenced by another portion of the 5′-UTR further upstream of the START codon. A deeper understanding of the role of the 5′-UTR in gene expression would improve criteria for choosing and using promoters inside yeast synthetic gene circuits. The online version of this article (doi:10.1186/s13036-017-0068-1) contains supplementary material, which is available to authorized users.
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影响因子：
3.7
作者：
Engler, Carola;Kandzia, Romy;Marillonnet, Sylvestre
通讯作者：
Marillonnet, Sylvestre
影响因子：
14.9
作者：
Gibson DG
通讯作者：
Gibson DG
影响因子：
64.5
作者：
GUARENTE, L;LALONDE, B;ALANI, E
通讯作者：
ALANI, E
DOI：
10.1073/pnas.1218066110
发表时间：
2013-02-19
影响因子：
11.1
作者：
Park, Chungoo;Chen, Xiaoshu;Zhang, Jianzhi
通讯作者：
Zhang, Jianzhi
影响因子：
14.9
作者：
ZUKER, M;STIEGLER, P
通讯作者：
STIEGLER, P