Exceptions to the Canonical Genetic Code in a Methanogenic Archaeon

从甲胺的甲烷生成（甲烷的合成）在微生物中由三种甲胺甲基转移酶之一启动，所述甲胺甲基转移酶特异性地使用三甲胺（TMA）、二甲胺（DMA）或单甲胺（MMA）甲基化类咕啉辅因子。编码TMA、DMA和MMA甲基转移酶的基因是非同源的，但每个基因含有单个框内琥珀密码子。如果琥珀密码子被读取为终止密码子，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显著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显示，通读发生没有移码，琥珀密码子被翻译为赖氨酸在一个代表性的甲基转移酶。转录物中没有明显的编辑或变化，表明琥珀密码子在赖氨酸发生时直接翻译。在巴氏甲烷八叠球菌（Methanosarcina barkeri）中发现了两个遗传学上不同的lys-tRNA合成酶（lysRS）基因。发现I类lysRS合成酶位于具有两个含琥珀甲基转移酶基因的操纵子下游，并且与MMA甲基转移酶基因共调节。组成型II类合成酶也在基因组中编码。I类酶的一个可能的作用是装载琥珀lys-tRNA，用于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。琥珀密码子通读的调节以及琥珀翻译的顺式要求将使用体外琥珀通读测定或替代地使用琥珀翻译的体内报告基因来检查。将构建突变菌株，其中甲胺甲基转移酶基因的琥珀密码子改变为典型的赖氨酸密码子，并测定对转录物、蛋白质水平和酶活性的影响。琥珀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，以表征它们对赖氨酸和不同lys-tRNA的行为。这些研究将揭示在这些产生甲烷的微生物中发现的通常遗传密码变化的功能。在一组特定的基因中，一个信号通常会停止蛋白质的生物合成，而不是指导氨基酸插入蛋白质。由于遗传密码是普遍存在的，因此遗传密码中的这种变化相对罕见。这项研究项目将部分解释这种遗传密码变化背后的生物驱动力。这有望为研究产甲烷微生物的蛋白质生物合成过程、基因调控和酶的催化活性提供新的见解。 了解这些过程很重要，因为甲烷生成是全球碳循环的重要组成部分。

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.