Exceptions to the Canonical Genetic Code in a Methanogenic archaeon

甲烷生产是研究产甲烷细菌的最初动力。 当前能源供应的固有脆弱性是继续研究的必要性。 产甲烷菌还占据了一个关键的生态位，防止碳在缺氧环境中积累。 污水消化等长期但实用的过程严重依赖产甲烷菌，许多有毒化学品在产甲烷条件下降解。 甲胺是产甲烷菌的优选底物。甲基-CoM是甲烷的倒数第二中间体。该实验室已经在甲基营养型甲烷菌巴氏甲烷八叠球菌（Methanosarcina barkeri）中从三甲胺（TMA）和一甲胺（MMA）生化重建了CoM甲基化的途径。这些途径共享相同的CoM甲基化酶蛋白，其与两种不同但同源的、对TMA或MMA具有特异性的类咕啉结合蛋白相互作用。每个途径的关键酶通过用MMA或TMA特异性甲基化类咕啉蛋白来启动代谢。TMA特异性甲基转移酶（TMAMT）由mttB编码。MMA特异性甲基转移酶（MMAMT）由mtmB编码。这些功能类似的酶几乎没有序列相似性，除了两个基因都含有符合读框的UAG密码子。如果用作终止密码子，框内UAG将导致比分离的甲基转移酶所观察到的短得多的蛋白质。UAG密码子是两种蛋白质的适当大小的开放阅读框的单一中断。对于mtmB，序列、阅读码框和基因分配已被彻底证实。UAG指导转录终止的能力在甲基转移酶的合成过程中明显被规避。这两种转录本都被预测在UAG密码子周围具有保守的结构和序列元件。UAG在M中作为终止密码子的频率非常低。巴克里。在获得将甲胺转化为甲烷的能力的过程中，甲烷八叠球菌似乎也获得了违反规范遗传密码的能力。一些假设的手段，防止终止在UAG是可能的，并将进行测试。将生成MMAMT的肽图，并通过质谱和Edman降解测定含UAG区域编码的肽的蛋白质序列。将表征由UAG编码的任何氨基酸。初步数据表明UAG通读是一个翻译事件，但转录本将进一步检查加工或编辑的证据。将在不同生长条件下使用全长蛋白的抗体测试UAG通读的效率。将比较来自相关属的不同mtmB和mttB基因，以确定UAG密码子的保守性、其位置和周围的序列/结构背景。将与含有5' UAG的区域的报告融合物引入染色体中，以确定序列背景和生长条件对UAG通读的影响。还将使用体外翻译系统来测试序列背景和生长条件对构建体中UAG通读的影响，所述构建体的范围从具有单个框内终止的短消息到含有全长mtmB的转录物。即使目前关于UAG定向终止抑制如何发生的模型是误导性的，仍然有证据表明第一个例子是在蛋白质编码基因中的内含子，这是一个在任何原核生物中很少见到的实体。 在这里学习新东西的潜力很大。 无论规避UAG定向终止的机制是什么，这个系统都提供了罕见的洞察力，让我们了解生物体如何进化出新的代谢能力，甚至改变它们的遗传密码。

Methane production was the original impetus for research into methanogenic bacteria. The innate vulnerability of current energy supplies underlies the need for their continued study. Methanogens also occupy a crucial ecological niche, preventing carbon accumulation in their anoxic environments. Prosaic but pragmatic processes such as sewage digestion rely heavily on methanogens, and many toxic chemicals are degraded under methanogenic conditions. Methylamines are preferred substrates of methanogens. Methyl-CoM is the penultimate intermediate to methane. This laboratory has biochemically reconstituted the pathways of CoM methylation from trimethylamine (TMA) and monomethylamine (MMA) in the methylotrophic methanogen Methanosarcina barkeri. These pathways share an identical CoM methylase protein, which interacts with two different, but homologous, corrinoid binding proteins specific for either TMA or MMA. The key enzymes of each pathway initiate metabolism by specifically methylating the corrinoid proteins with MMA or TMA. The TMA specific methyltransferase (TMAMT) is encoded by mttB. The MMA specific methyltransferase (MMAMT) is encoded by mtmB. These functionally analogous enzymes share little sequence similarity, except that both genes contain in-frame UAG codons. If used as a stop codon, the in-frame UAG would result in much shorter proteins than observed for the isolated methyltransferases. The UAG codons are the single interruptions of otherwise open reading frames of proper size for both proteins. For mtmB, the sequence, reading frame, and gene assignment have been exhaustively confirmed. The ability of UAG to direct transcriptional termination is clearly circumvented during synthesis of the methyltransferases. Both transcripts are predicted to have conserved structure and sequence elements around the UAG codon. UAG is used at unusually low frequency as a stop codon in M. barkeri. It appears that during the acquisition of the ability to convert methylamines to methane Methanosarcina also acquired a violation of the canonical genetic code. A number of hypothetical means of preventing termination at UAG are possible and will be tested. Peptide maps of MMAMT will be generated and the protein sequence of peptides encoded by the UAG-containing regions will be determined by mass spectroscopy and Edman degradation. Any amino acid encoded by UAG will be characterized. Preliminary data indicates UAG readthrough is a translational event, but transcripts will be further examined for evidence of processing or editing. The efficiency of UAG readthrough will be tested under different growth conditions using antibody to the full-length protein. Divergent mtmB and mttB genes from related genera will be compared to ascertain conservation of the UAG codon, its position, and surrounding sequence/structure context. Reporter fusions with 5' UAG containing regions will be introduced into the chromosome in order to determine effects of sequence context and growth conditions on UAG readthrough. An in vitro translation system will also be used to test the effect of sequence context and growth conditions on UAG readthrough in constructs ranging from short messages with single in-frame stops to full length mtmB containing transcripts. Even if the current model for how suppression of UAG directed termination occurs is misleading, there will remain evidence for the first example of an intron in protein coding genes in the Archaea, an entity very seldom seen among any prokaryote. The potential to learn something new here is high. No matter what the mechanism for circumventing UAG-directed termination may turn out to be, this system offers rare insight into how organisms evolve new metabolic capabilities, even to the extent of modifying their genetic codes.