Reducing the genetic code induces massive rearrangement of the proteome
Reducing the genetic code induces massive rearrangement of the proteome
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DOI：
10.1073/pnas.1420193111
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发表时间：
2014-11
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影响因子：
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通讯作者：
P. O'Donoghue;Laure Prat;Martin Kucklick;Johannes G. Schäfer;K. Riedel;J. Rinehart;D. Söll;Ilka U. Heinemann
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文献类型：
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作者：
P. O'Donoghue;Laure Prat;Martin Kucklick;Johannes G. Schäfer;K. Riedel;J. Rinehart;D. Söll;Ilka U. Heinemann
扩展遗传密码是合成生物学的一个重要目标，但一些生物在进化过程中自然地发展了扩展的遗传密码。为了了解遗传编码超过20个氨基酸的选择性优势，我们研究了将Methanosarcina acetivorans的遗传密码从21个氨基酸减少到20个氨基酸的蛋白质组范围内的反应。数据显示了天然蛋白质组如何适应遗传密码简化，并表明扩展遗传密码的选择价值与碳源范围和代谢效率相关。扩展遗传密码是合成生物学的一个重要目标，但一些生物在进化过程中很久以前就发展出了自然扩展的遗传密码。所有测序的基因组中不到1%编码将终止密码子UAG重新指定为吡咯赖氨酸（Pyl）的操纵子，这是由Pyl-tRNAPyl的生物合成产生的遗传密码变体。为了理解遗传编码超过20个氨基酸的选择性优势，我们构建了不能将UAG解码为Pyl或在三甲基胺上生长的无标记tRNAPyl缺失的乙酸甲烷八叠球菌菌株（ΔpylT）。在含有甲醇的基本培养基中，ΔpylT菌株的表型缺陷是明显的。野生型（WT）M.乙酸菌和ΔpylT细胞从MS/MS检测到的> 7，000个显著肽中鉴定出841个蛋白质。通过MS/MS验证八种蛋白质的UAG密码子的翻译，包括鉴定PylB中的Pyl残基，其催化Pyl生物合成的第一步。tRNAPyl的缺失全面改变了蛋白质组，导致>300种差异丰富的蛋白质。将遗传密码从21个氨基酸减少到20个氨基酸导致翻译起始因子、氨基酸代谢和甲醇产甲烷的显著下调，这被二甲硫醚代谢酶的补偿性（100倍）上调所抵消。这些数据显示了天然蛋白质组如何适应遗传密码简化，并表明扩展的遗传密码的选择价值与碳源范围和代谢效率有关。
Significance Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes over the course of evolution. To understand the selective advantage of genetically encoding more than 20 amino acids, we investigated the proteome-wide response to reducing the genetic code of Methanosarcina acetivorans from 21 to 20 amino acids. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency. Expanding the genetic code is an important aim of synthetic biology, but some organisms developed naturally expanded genetic codes long ago over the course of evolution. Less than 1% of all sequenced genomes encode an operon that reassigns the stop codon UAG to pyrrolysine (Pyl), a genetic code variant that results from the biosynthesis of Pyl-tRNAPyl. To understand the selective advantage of genetically encoding more than 20 amino acids, we constructed a markerless tRNAPyl deletion strain of Methanosarcina acetivorans (ΔpylT) that cannot decode UAG as Pyl or grow on trimethylamine. Phenotypic defects in the ΔpylT strain were evident in minimal medium containing methanol. Proteomic analyses of wild type (WT) M. acetivorans and ΔpylT cells identified 841 proteins from >7,000 significant peptides detected by MS/MS. Protein production from UAG-containing mRNAs was verified for 19 proteins. Translation of UAG codons was verified by MS/MS for eight proteins, including identification of a Pyl residue in PylB, which catalyzes the first step of Pyl biosynthesis. Deletion of tRNAPyl globally altered the proteome, leading to >300 differentially abundant proteins. Reduction of the genetic code from 21 to 20 amino acids led to significant down-regulation in translation initiation factors, amino acid metabolism, and methanogenesis from methanol, which was offset by a compensatory (100-fold) up-regulation in dimethyl sulfide metabolic enzymes. The data show how a natural proteome adapts to genetic code reduction and indicate that the selective value of an expanded genetic code is related to carbon source range and metabolic efficiency.