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: 9808914
• 负责人: Joseph Krzycki
• 金额: --
• 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: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9808914&HistoricalAwards=false
• 关键词: Exceptions; Canonical; Genetic; Code; Methanogenic

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.

甲烷的产生是研究产甲烷细菌的最初动力。当前能源供应的先天脆弱性是对其继续研究的基础。产甲烷菌也占据了一个至关重要的生态位，阻止了它们在缺氧环境中的碳积累。诸如污水消化等平凡但实用的过程严重依赖于产甲烷菌，许多有毒化学物质在产甲烷条件下被降解。甲胺是产甲烷菌的首选底物。甲基com是甲烷的倒数第二中间体。本实验室对甲基化产甲烷菌barkeri中三甲胺（TMA）和一甲基胺（MMA）的CoM甲基化途径进行了生化重建。这些途径共享一个相同的CoM甲基化酶蛋白，该蛋白与两种不同但同源的蛋白相互作用，特异性针对TMA或MMA。每条通路的关键酶通过特异性地甲基化含MMA或TMA的类玉米粉蛋白来启动代谢。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。除了两个基因都含有帧内UAG密码子外，这些功能类似的酶几乎没有序列相似性。如果作为终止密码子使用，则框架内的UAG将导致比分离的甲基转移酶所观察到的更短的蛋白质。UAG密码子是两种蛋白质正常大小的开放阅读框的单个中断。对于mtmB，序列、阅读框和基因分配已经得到了详尽的确认。在甲基转移酶的合成过程中，UAG直接转录终止的能力显然被规避了。预计这两个转录本在UAG密码子周围具有保守的结构和序列元件。在巴氏分枝杆菌中，UAG作为终止密码子的使用频率异常低。看来，在获得将甲胺转化为甲烷的能力的过程中，甲烷藻也获得了对典型遗传密码的违反。一些假设的防止在UAG终止的方法是可能的，并将进行测试。生成MMAMT的肽图，并通过质谱和Edman降解确定含有uag区域编码的肽的蛋白质序列。任何被UAG编码的氨基酸都将被表征。初步数据表明，UAG读通是一个翻译事件，但转录本将进一步检查处理或编辑的证据。UAG的读取效率将在不同的生长条件下使用抗体对全长蛋白进行测试。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、位置和周围的序列/结构背景。将含有5' UAG区域的报告融合物引入染色体，以确定序列背景和生长条件对UAG读透的影响。体外翻译系统还将用于测试序列上下文和生长条件对UAG读通的影响，从具有单个帧内停止的短消息到包含转录本的全长mtmB。即使目前关于抑制UAG定向终止如何发生的模型是误导性的，仍然有证据表明，在古生菌的蛋白质编码基因中存在第一个内含子，这在任何原核生物中都很少见。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，该系统都为生物体如何进化出新的代谢能力，甚至在修改其遗传密码的程度上提供了难得的见解。