MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

该建议涉及+1翻译移码的机制， 这两种事件都发生在特殊的mRNA位点， 阅读框架（程序性移码）和表型抑制 由突变型tRNA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没有关联 机械地。 移码抑制的经典模型 提出了一种具有扩展的反密码子环的突变tRNA， 扩展了四个核苷酸的反密码子，导致下游的转移， 或者+1方向。 相比之下，程序化的移码现在似乎发生了 因为最后一个框内密码子的近同源解码， 到下一个密码子移位阅读时的翻译错误。 的 经典的抑制模型现在看来是无效的，抑制可能 通过类似于编程移码的机制发生。 这项建议 寻求进一步探索程序化框架转换的机制， 为了测试用于移码的近同源解码模型是否 压制是正确的。 酵母分子遗传学分析将用于进一步测试 近同源解码模型的有效性 移码 基于PCR的基因破坏将消除每一个 酵母基因组中的低拷贝tRNA基因;在这些菌株中， 密码子必须由近同源的同种受体读取，这可能诱导高表达。 效率框架转换 在某些情况下，发生移码是因为 正常的同源tRNA不能有效地解码doo;位点特异性 诱变将鉴定tRNA中有助于 他们的低效率。 一种促进+1的体外翻译系统 编程移码将用于识别哪些近同源 tRNA实际上负责促进框架的转变。 最后，顺式作用mRNA的移码刺激的基础 序列，并通过延伸因子-1 α的突变体进行评估 使用位点特异性诱变和经典遗传分析。 一种新的移码表型抑制模型的关键特征 将在提案中测试突变。 第一，需要一个 tRNA和mRNA之间的四核苷酸相互作用将通过以下方法进行测试： 编码移码抑制子基因的位点特异性诱变 tRNA。 第二，该提案将测试预测， 反密码子环稳定近同源解码， 同源解码 第三，它将检验扩张是否只会刺激 由可以在mRNA上滑动的tRNA引起的移码。 最后将 测试是否有相同的机制控制-1移码的抑制 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.