MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

这一建议涉及+1平移框架的机制， 这两个事件都发生在特殊的mRNA位点上，它们促进了 阅读框架(程控移码)与表型抑制 突变tRNAs引起的移码突变(移码抑制)。 长期以来，人们一直认为这两件事毫无关联 从机械上讲。帧移位抑制的经典模型 建议具有扩展反密码子环的突变体tRNA读取 扩展了四个核苷酸的反密码子，导致下游的移位 或+1方向。相比之下，程序化的框架移动现在似乎发生了 因为对最后一个帧内密码子进行了近似同源的解码，这导致 到下一次密码子移位阅读时的翻译错误。这个 经典的抑制模型现在似乎是无效的，并且抑制可能 发生的机制类似于程控移帧。这项建议 旨在进一步探索程序化移帧的机制和 测试帧移位的近同源解码模型 压制是正确的。 酵母分子遗传分析将被用来进一步测试 近同源译码模型在程序设计中的有效性 帧移位。基于聚合酶链式反应的基因破坏将消除每个 酵母基因组中的低拷贝tRNA基因；在这种菌株中，同源基因 密码子必须由接近同源的同源同源受体阅读，这可能导致高 效率移帧。在某些情况下，发生帧移位是因为 正常同源tRNA对DOO的解码效率低下；位点特异性 诱变将确定tRNA中有助于 他们的效率低下。一种促进+1的体外翻译系统 程序移码将被用来识别哪个近亲 TRNA实际上负责促进框架的转变。 最后，顺式作用信使核糖核酸刺激移码的基础 序列，以及通过突变的延伸因子-1α将被评估 使用定点突变和经典遗传分析。 一种新的移码表型抑制模型的主要特征 突变将在提案中进行测试。首先，有必要建立一个 TRNA和mRNA之间的四个核苷酸相互作用将通过以下方式进行测试 编码移码抑制基因的定点突变 TRNA。其次，该提案将考验经济扩张的预测。 反密码子环稳定近同源解码，并破坏稳定 同源译码。第三，它将测试扩张是否只会刺激 通过tRNAs进行移码，这可能会滑落到mRNA上。最后，它将 测试相同的机制是否控制抑制-1\f25 FrameShift-1 突变。

This proposal concerns the mechanism of +1 translational frameshifting, both events which occur at special mRNA sites which promote a shift in reading frame (programmed frameshifting) and phenotypic suppression of frameshift mutations caused by mutant tRNAs (frameshift suppression). It has long been thought that these two events were unrelated mechanistically. The classical model for frameshift suppression proposes that a mutant tRNA with an expanded anticodon loop reads an expanded four nucleotide anticodon, causing a shift in the downstream or +1 direction. Programmed frameshifts by contrast now appear to occur because of near-cognate decoding of the last in-frame codon, which leads to a translational error at the next codon shifting reading. The classical model of suppression now appears invalid, and suppression may occur by a mechanism similar to programmed frameshifts. This proposal seeks to further explore the mechanism of programmed frameshifting and to test whether the near-cognate decoding model for frameshift suppression is correct. Yeast molecular genetic analysis will be used to test further the validity of the near-cognate decoding model for programmed frameshifting. PCR-based gene disruption will eliminate each of the low-copy tRNA genes in the yeast genome; in such strains the cognate codons must be read by a near-cognate isoacceptor, which may induce high efficiency frameshifting. In some cases frameshifting occurs because the normal cognate tRNA decodes doo inefficiently; site-specific mutagenesis will identify the sequences in the tRNAs contributing to their inefficiency. An in vitro translation system which promotes +1 programmed frameshifting will be used to identify which near-cognate tRNAs are actually responsible for promoting the shift in frame. Finally, the basis of frameshift stimulation by a cis-acting mRNA sequence, and by mutants of elongation factor-1alpha will be assessed using site-specific mutagenesis and classical genetic analysis. Key features of a new model for phenotypic suppression of frameshift mutations will be tested in the proposal. First, the necessity for a four nucleotide interaction between the tRNA and mRNA will be tested by site-specific mutagenesis of a gene encodig a frameshift suppressor tRNA. Second, the proposal will test the prediction that expansion of the anticodon loop stabilizes near-cognate decoding, and destabilizes cognate decoding. Third, it will test whether expansion only stimulates frameshifting by tRNAs which can slip on the mRNA. Finally, it will test if the same mechanism governs suppression of -1 frameshift mutations.