MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

酵母翻译记录的分子基础

• 批准号: 2391900
• 负责人: Philip James Farabaugh
• 金额: $ 24.97万
• 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 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2391900
• 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; molecular cloning; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; point mutation; ribosomes; transposon /insertion element

The ribosome is a molecular machine which translates the information encoded in a cell's genome into protein products. It has the twin roles of providing an accurate representation of that information and producing the product rapidly. These roles are fundamentally in conflict since to the extent that translational accuracy increases, the rate of translation decreases. Even so, the ribosome achieves a high degree of accuracy, with an error rate estimated at less than 5 X 10(-4) per codon. How is this very high accuracy achieved? One way of addressing the mechanism of translational accuracy is to consider how specific sequences may perturb it. Programmed frameshift sites are regions of mRNAs which cause efficient changes in reading frame either shifting to the 3' (negative frameshifting) or 5' (positive frameshifting). We would like to understand how one such site which induces +1 frameshifting manipulates the translational apparatus. The retrotransposon Ty3 encodes the product of the POL3 gene as a translational fusion to the upstream GAG3 gene. We have already demonstrated that the event occurs by + l frameshifting within a sequence GCG-AGU-U (shown as codons of GAG3). We have also identified all possible substitutes for the GCG and AGU-U codons. We would like to understand how the frameshift is stimulated. First, we will determine how many 7 nt +1 frameshift sites there are by random oligonucleotide mutagenesis. Second, the tRNA decoding GCG appears to be special in its ability to stimulate frameshifting without itself slipping on the mRNA template. We will attempt to determine what features of this, and other, "P- site" tRNAs stimulate frameshifting. The "A-site" tRNA decoding the first +i frame codon, GUU may also be special in driving frameshifting into the +1 frame; we will test this hypothesis by overexpressing and mutagenizing the tRNA. Ty3 frameshifting is stimulated by a downstream "context", though we don't know how. Some of the hypotheses we will test is that the nascent protein product of the context perturbs frameshifting, or that the context, as RNA, interacts with some element of the translational machinery (elongation factor, ribosomal protein or ribosomal RNA). Finally, we will look for interactions between specific A and P-site tRNAs and other components of the translational machinery to identify trans-acting factors essential to frameshifting. These studies will provide an intellectual basis for understanding the ways in which programmed frameshift sites interact with the translational machinery. The results of these studies will be relevant to our understanding how the ribosome, as a molecular machine, functions to rapidly and accurately decode the genetic information.

核糖体是翻译信息的分子机器 在细胞基因组中编码成蛋白质产物。它具有双重作用： 提供该信息的准确表示并生成 产品迅速。这些角色从根本上来说是冲突的 翻译准确性提高的程度，翻译率 减少。即便如此，核糖体仍然达到了很高的准确性， 每个密码子的错误率估计小于 5 X 10(-4)。这是怎么样 达到非常高的准确度？解决机制的一种方法 翻译准确性是考虑特定序列如何干扰 它。程序化移码位点是 mRNA 的区域，可导致有效的 阅读框的变化要么转移到 3'（负 移码）或 5'（正移码）。我们想了解 一个这样的引发+1移码的位点如何操纵 翻译装置。 反转录转座子 Ty3 将 POL3 基因的产物编码为 与上游 GAG3 基因的翻译融合。我们已经 证明该事件是通过序列内的 + l 移码发生的 GCG-AGU-U（显示为GAG3的密码子）。我们还确定了所有可能的 替代 GCG 和 AGU-U 密码子。我们想了解如何 移码受到刺激。首先，我们要确定有多少个7 nt +1 通过随机寡核苷酸诱变产生移码位点。第二， tRNA 解码 GCG 的特殊之处在于它能够刺激 移码，而自身不会在 mRNA 模板上滑动。我们将 尝试确定该 tRNA 和其他“P 位点”tRNA 的特征 刺激移码。 “A位点”tRNA解码第一个+i帧 密码子，GUU也可能在驱动移码到+1框架中是特殊的； 我们将通过过度表达和诱变 tRNA 来检验这一假设。 Ty3 移码是由下游“环境”刺激的，尽管我们不这样做 知识。我们将测试的一些假设是新生蛋白质 上下文的产物扰乱移码，或者上下文，如 RNA，与翻译机器的某些元件相互作用 （延伸因子、核糖体蛋白或核糖体RNA）。最后，我们将 寻找特定 A 和 P 位点 tRNA 与其他 转化机制的组成部分来识别反式作用因子 对于移码至关重要。 这些研究将为理解 编程移码位点与翻译相互作用的方式 机械。这些研究的结果将与我们相关 了解核糖体作为分子机器如何发挥作用 快速准确地解码遗传信息。