Translation Recoding Control

“重新编码”是一种非标准的遗传解码，比如翻译移码。抗酶是多胺的一种调节蛋白，它的表达和多胺的诱导需要翻译移码。我们已经确定了AZ的主要亚型AZ1的移码信号序列，即移位位置、下游伪结和上游序列。本课题不仅从顺式作用信号的角度，而且从翻译机制和小分子调节子的角度来研究AZ1的移码机制，主要研究结果如下：(1)分析了AZ1移码信号的分子进化、与其他编码信号的互换性以及多胺作用的作用部位。AZ1假结的细胞结合蛋白已被鉴定。一些与移码信号互补结合的小核苷酸被发现影响移码效率。(2)在由哺乳动物AZL序列在大肠杆菌中进行的核糖体跳跃中，肽基-tRNA的重新配对被发现是着陆密码子的主要决定因素。(3)在裂解酵母中进行遗传筛选，以确定参与AZ1移码的翻译机制。我们筛选了基因组突变、多拷贝抑制因子和显性抑制因子，但未能获得任何有意义的候选基因。(4)嗜热菌来源的某些支链多胺或乙酰化形式的多胺对AZ1移码有特异性作用。(5)为了发现新的编码基因，我们在大肠杆菌中筛选了一个随机序列池。我们还搜索了裂解酵母中无义介导的mRNA衰变突变体中表达水平升高的mRNAs。这两次筛查都带来了几个新的重新编码候选者。(6)对AZ1基因敲除小鼠的分析表明，AZL缺乏移码调控的表型是由于造血细胞分化障碍导致的胚胎死亡。

"Recoding" is non-standard genetic decoding such as translational frameshifting. Antizyme, a regulatory protein for polyamines, requires translational frameshifting for its expression and induction by polyamines. We have identified the signal sequences, namely, the shift site, a downstream pseudoknot, and an upstream sequence, for the frameshifting of AZ1, an major isoform of AZ. This project aimed to understand the mechanism of AZ1 frameshifting form the aspects of not only the cis-acting signals but also translational machinery and small-molecule regulators, and the results are summarized as the followings. (1) The molecular evolution, interchangeability with other recoding signals, and the interaction site of the polyamine action of the AZ1 frameshift signal were analyzed. A cellular binding protein for AZ1 pseudoknot has been identified. Some small nucleotides that complementarily bind to the frameshift signals are found to affect frameshift efficiency. (2) In the ribosomal hopping directed by the mammalian AZl sequence in E. coli, re-pairing of the peptidyl-tRNA is found to be major determinant of the landing codon. (3) To identify translation machinery involved in AZ1 frameshifting, genetic screening was performed in the fission yeast. We screened genomic mutations, multicopy suppressors, dominant suppressors, but could not obtain any significant candidate. (4) Some branched-chain polyamines originated from a thermophilic bacteria or acetylated forms of polyamines showed specific effect on AZ1 frameshifting. (5) To discover new recoding genes, we screed a random sequence pool in E. coli. We also searched mRNAs of which expression levels are elevated in the nonsense-mediated mRNA decay mutants in the fission yeast. Both screening brought about several candidate for new recoding. (6) Analysis of AZ1 knockout mice revealed that a phenotype of lack of frameshift regulation of AZl is embryonic death due to a disturbance of hematopoietic cell differentiation.