Translation Recoding Control

“重编码”是指非标准的基因解码，如平移移帧。抗酶是一种多胺调节蛋白，其表达和诱导需要翻译移框。我们已经确定了AZ1 （AZ的主要同工异构体）移框的信号序列，即移位位点、下游伪结和上游序列。本项目旨在从顺式作用信号、翻译机制和小分子调控因子等方面了解AZ1移框的机制，并将结果总结如下。(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相互作用位点。已鉴定出AZ1假结的细胞结合蛋白。发现一些与移码信号互补结合的小核苷酸会影响移码效率。(2)在大肠杆菌中由哺乳动物AZl序列引导的核糖体跳跃中，发现肽基trna的重新配对是着陆密码子的主要决定因素。(3)为了确定AZ1移框过程中涉及的翻译机制，对裂变酵母进行了遗传筛选。我们筛选了基因组突变、多拷贝抑制子、显性抑制子，但没有获得任何显著的候选基因。(4)一些源自嗜热细菌的支链多胺或多胺的乙酰化形式对AZ1移框有特异性影响。(5)为了发现新的编码基因，我们在大肠杆菌中随机筛选了一个序列池。我们还在裂变酵母中无义介导的mRNA衰变突变体中寻找表达水平升高的mRNA。这两种筛选都产生了一些新的重编码候选基因。(6)对AZ1基因敲除小鼠的分析表明，AZl基因移码调控缺失的表型是由于造血细胞分化受阻导致的胚胎死亡。

"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.