MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

酵母翻译记录的分子基础

• 批准号: 2900549
• 负责人: Philip James Farabaugh
• 金额: $ 30.03万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE COUNTY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900549
• 关键词: RNA splicing; Saccharomyces; endonuclease; frameshift mutation; fungal genetics; gel electrophoresis; gene expression; genetic mapping; genetic promoter element; genetic recombination; genetic transcription; genetic translation; mass spectrometry; molecular cloning; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; polymerase chain reaction; ribosomes; site directed mutagenesis; transposon /insertion element

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.

该提案涉及+1平移移码机制， 这两个事件都发生在促进转变的特殊 mRNA 位点上 阅读框（程序移码）和表型抑制 由突变 tRNA 引起的移码突变（移码抑制）。 长期以来人们一直认为这两起事件无关 机械地。 移码抑制的经典模型 提出具有扩展反密码子环的突变 tRNA 读取 扩展了四个核苷酸反密码子，导致下游的转变 或+1方向。 相比之下，现在似乎发生了编程移码 由于最后一个帧内密码子的近同源解码，这导致 到下一个密码子转换阅读时的翻译错误。 这 经典的抑制模型现在看来无效，并且抑制可能 通过类似于编程移码的机制发生。 这个提议 旨在进一步探索程序性移码机制 测试近同源解码模型是否适用于移码 压制是正确的。 酵母分子遗传学分析将用于进一步测试 程序的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；在这样的菌株中同源 密码子必须由近同源的同源受体读取，这可能会导致高 效率移码。 在某些情况下，发生移码是因为 正常同源 tRNA 解码 doo 的效率低下；特定地点的 诱变将鉴定 tRNA 中有助于 他们的低效率。 促进+1的体外翻译系统 程序化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 刺激移码的基础 序列，并通过延伸因子-1α的突变体进行评估 使用位点特异性诱变和经典遗传分析。 移码表型抑制新模型的主要特征 提案中将测试突变。 首先，需要一个 tRNA 和 mRNA 之间的四个核苷酸相互作用将通过 编码移码抑制子的基因的定点诱变 tRNA。 其次，该提案将检验扩大规模的预测。 反密码子循环稳定近同源解码，并不稳定 同源解码。 第三，检验扩张是否只是刺激 tRNA 可以在 mRNA 上滑动，从而实现移码。 最后，它将 测试是否相同的机制控制 -1 移码抑制 突变。