Pyrrolysine, a novel genetically encoded amino acid

描述(申请人提供)：吡咯赖氨酸，一种新的氨基酸，最近被发现由甲胺代谢的微生物基因中的UAG密码子编码。这是自18年前发现硒半胱氨酸由微生物中的UGA密码子编码以来，第二个由基因编码的非规范氨基酸的例子。现已知，硒半胱氨酸在人体内分布广泛，是硒的主要形式。遵循这一先例，吡咯赖氨酸有可能在新陈代谢中更广泛地分布和输入。无论如何，吡咯赖氨酸将成为生物体如何调节其遗传密码以扩大新陈代谢能力的罕见例子，这一成就的复制有望人工定制具有生物医学潜力的新蛋白质。我们的长期目标是了解吡咯赖氨酸的功能、生物发生和遗传编码。UAG密码子的翻译被认为需要一个专门的UAG解码tRNA和一个专门的同源氨基酰-tRNA合成酶。这些基因产物和其他被认为参与将UAG解码为吡咯赖氨酸的基因产物的作用将使用生化和遗传学方法进行研究。吡咯赖氨酸的最终结构尚不清楚，通过分析蛋白质、胞浆和tRNA中的UAG编码残基，可以深入了解吡咯赖氨酸的结构和遗传编码。已经开发出一种体内系统，将用于研究UAG在将其翻译为吡咯赖氨酸过程中如何作为正义而不是终止密码子发挥作用。一个类似的系统将被用于定点突变，以测试吡咯赖氨酸在酶催化中的假设功能。最后，转座子突变将被用来鉴定在UAG翻译为吡咯赖氨酸过程中具有预期或未预期功能的未知基因。

DESCRIPTION (provided by applicant): Pyrrolysine, a novel amino acid, was recently found to be encoded by UAG codons in a microbial gene for methylamine metabolism. This is the second example of a genetically encoded non-canonical amino acid since selenocysteine, which 18 years ago was found to be encoded by UGA codons in a microbe. Selenocysteine is now known to be widely distributed and the primary form of selenium in humans. Following this precedent, pyrrolysine holds potential of being more widely distributed and of import in metabolism. Regardless, pyrrolysine will serve as a rare example of how organisms can modulate their genetic code to expand metabolic capabilities, an achievement whose replication holds promise for artificially tailoring novel proteins with biomedical potential. Our long-range goals are to understand the function, biogenesis, and genetic encoding of pyrrolysine. Translation of UAG codons is hypothesized to require a specialized UAG decoding tRNA and a dedicated cognate aminoacyl-tRNA synthetase. The roles of these gene products and others thought to be involved in UAG decoding as pyrrolysine will be investigated using biochemical and genetic approaches. The final structure of pyrrolysine is as yet unknown, and insight into pyrrolysine structure and genetic encoding will be gained by analyzing the UAG encoded residue in protein, the cytosol, and on tRNA. An in vivo system has been developed that will be used to study how UAG functions as a sense rather than a stop codon during UAG translation as pyrrolysine. A similar system will be used for site directed mutagenesis in order to test the hypothesized function of pyrrolysine in enzyme catalysis. Finally, transposon mutagenesis will be used to identify unknown genes with anticipated or unanticipated functions during UAG translation as pyrrolysine.