Exceptions to the Canonical Genetic Code in a Methanogenic Archaeon

产甲烷古菌中规范遗传密码的例外

• 批准号: 0114797
• 负责人: Joseph Krzycki
• 金额: $ 37万
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0114797&HistoricalAwards=false
• 关键词: Exceptions; Canonical; Genetic; Code; Methanogenic

Methanogenesis (the synthesis of methane) from methylamines is initiated in microbes by one of three methylamine methyltransferases that methylate corrinoid cofactors specifically using either trimethylamine (TMA), dimethylamine (DMA), or monomethylamine (MMA). The genes encoding the TMA, DMA, and MMA methyltransferases are non-homologous, yet each gene contains a single in-frame amber codon. If amber codons were read as stop codons, each methylamine methyltransferase gene would produce an amber termination product markedly truncated relative to the isolated and characterized methyltransferase. Sequencing of the tryptic fragments of the MMA methyltransferase revealed that readthrough occurs without a frameshift, and that the amber codon is translated as lysine in a representative methyltransferase. No editing or changes are evident in transcripts, indicating direct translation of the amber codon as lysine occurs. Two phylogenetically distinct lys-tRNA synthetase (lysRS) genes were found in Methanosarcina barkeri. A class I lysRS synthetase is found downstream of an operon with two of the amber containing methyltransferase genes and is co-regulated with the MMA methyltransferase gene. A constitutive class II synthetase is also encoded in the genome. A possible role of the class I enzyme is charging an amber lys-tRNA for translation of amber as lysine. Working hypotheses are that the amber codons are required for either proper folding and/or translational processing of the methyltransferase proteins, or are involved in controlling methyltransferase transcription and translation. The regulation of amber codon readthrough, as well as cis requirements for amber translation, will be examined using either an in vitro amber readthrough assay, or alternatively, using in vivo reporters of amber translation. A mutant strain will be constructed in which the amber codon of a methylamine methyltransferase gene is changed to a canonical lysine codon, and the effect on transcripts, protein levels and enzyme activity determined. The amber lys-tRNA will be identified by one of several alternate approaches and characterized. The two lysRS enzymes will either be expressed recombinantly or isolated from the native host in order to characterize their behavior with respect to lysine and different lys-tRNAs. These studies will shed light on the function of a change in the usual genetic code found in these methane producing microorganisms. In a select group of their genes a signal that usually stops biosynthesis of a protein, instead directs the insertion of an amino acid into the protein. Since the genetic code is universal such changes in the genetic code are relatively rare. This research project will in part explain the biological driving force behind this change in the genetic code. This is expected to provide new insights into the process of protein biosynthesis, gene regulation, and the catalytic activities of enzymes in methane-producing microbes. Understanding these processes is important because methanogenesis is an essential component of the global carbon cycle.

甲胺的产甲烷作用（甲烷的合成）是由微生物中的三种甲胺甲基转移酶之一启动的，这些酶专门使用三甲胺 (TMA)、二甲胺 (DMA) 或一甲胺 (MMA) 将类咕啉辅助因子甲基化。编码 TMA、DMA 和 MMA 甲基转移酶的基因是非同源的，但每个基因都包含一个框内琥珀密码子。如果将琥珀密码子解读为终止密码子，则每个甲胺甲基转移酶基因将产生相对于分离和表征的甲基转移酶明显截短的琥珀终止产物。 MMA 甲基转移酶的胰蛋白酶片段的测序表明，通读发生时没有移码，并且琥珀密码子在代表性甲基转移酶中被翻译为赖氨酸。转录本中没有明显的编辑或变化，表明琥珀密码子直接翻译为赖氨酸。在巴克甲烷八叠球菌中发现了两个系统发育上不同的 lys-tRNA 合成酶 (lysRS) 基因。 I 类 lysRS 合成酶位于操纵子下游，具有两个含有琥珀的甲基转移酶基因，并与 MMA 甲基转移酶基因共同调节。基因组中还编码了组成型 II 类合成酶。 I 类酶的一个可能作用是为琥珀色 lys-tRNA 充电，将琥珀翻译为赖氨酸。工作假设是琥珀密码子是甲基转移酶蛋白的正确折叠和/或翻译加工所必需的，或者参与控制甲基转移酶转录和翻译。将使用体外琥珀通读测定或使用琥珀翻译的体内报告基因来检查琥珀密码子通读的调节以及琥珀翻译的顺式要求。将构建突变菌株，其中甲胺甲基转移酶基因的琥珀密码子被改变为规范的赖氨酸密码子，并确定对转录物、蛋白质水平和酶活性的影响。琥珀色 lys-tRNA 将通过几种替代方法之一进行鉴定和表征。这两种 lysRS 酶将重组表达或从天然宿主中分离，以便表征它们对于赖氨酸和不同 lys-tRNA 的行为。这些研究将揭示这些产甲烷微生物中常见遗传密码变化的功能。在一组选定的基因中，信号通常会停止蛋白质的生物合成，而是引导氨基酸插入蛋白质中。由于遗传密码是通用的，遗传密码的这种变化相对罕见。该研究项目将部分解释遗传密码变化背后的生物驱动力。这有望为蛋白质生物合成过程、基因调控以及产甲烷微生物中酶的催化活性提供新的见解。 了解这些过程很重要，因为产甲烷作用是全球碳循环的重要组成部分。