MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

核糖体是翻译信息的分子机器 编码在细胞基因组中的蛋白质产物。它具有双重作用，即 提供该信息的准确表示，并生成 产品生产速度快。这些角色从根本上是冲突的，因为 翻译准确率提高的程度，翻译的速度 减少。即使如此，核糖体也达到了高度的准确性， 估计每个密码子的错误率小于5×10(-4)。这是怎么回事 达到了非常高的精确度？一种解决机制的方法是 翻译的准确性是考虑特定序列可能如何干扰 它。程序性移码位点是mRNAs的区域，可导致高效 阅读框的变化或者移动到3‘(负数 移码)或5‘(正移码)。我们想要了解 这样一个导致移帧的站点是如何操纵 翻译仪器。 反转录转座子Ty3编码POL3基因的产物为 与上游GAG3基因的翻译融合。我们已经这么做了 演示了事件是由L在一个序列内进行的移帧发生的 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。我们想要了解如何 帧转换受到了刺激。首先，我们将确定有多少7个新台币1 移码位点存在由随机寡核苷酸引起的突变。第二, TRNA解码GCG的特殊之处在于它的刺激能力 在信使核糖核酸模板上无自身滑动的情况下进行移码。我们会 尝试确定此tRNA和其他“P-Site”tRNA的哪些功能 刺激移帧。“A位”tRNA对第一个I帧的解码 密码子、GUU也可能是驱动移码到第1帧的特殊基因； 我们将通过过度表达和突变tRNA来检验这一假设。 Ty3的移帧受到下游“语境”的刺激，但我们不会 知道怎么做。我们将检验的一些假设是，新生蛋白质 上下文的乘积扰乱了帧移动，或者上下文，如 RNA，与翻译机制的某些元素相互作用 (延伸因子、核糖体蛋白或核糖体RNA)。最后，我们会 寻找特定A和P位点tRNA之间的相互作用以及其他 识别反式作用因子的翻译机制的组成部分 对移帧至关重要。 这些研究将为理解 编程的框架转换站点与翻译的 机械设备。这些研究的结果将与我们的 了解作为分子机器的核糖体是如何发挥作用的 快速、准确地解码遗传信息。

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.