MOLECULAR BASIS OF TRANSLATIONAL RECODING IN YEAST

核糖体是一种分子机器， 在细胞的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。这些角色从根本上是冲突的， 翻译准确性提高的程度，翻译的速度 减少。即便如此，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）。这怎么 实现了极高的精度？解决这一机制的一种方法是， 翻译准确性是考虑特定序列如何干扰 了程序化移码位点是mRNA的区域， 阅读帧中的变化或者移位到3'（负 移码）或5 ′（正移码）。我们想要理解 一个诱导+1移码的位点是如何操纵 翻译器 逆转录转座子Ty 3编码POL 3基因的产物作为一种转录因子。 与上游GAG 3基因的翻译融合。我们已经 证明了事件是通过序列中的+ l移码发生的 GCG-AGU-U（显示为GAG 3的密码子）。我们还确定了所有可能的 GCG和AGU-U密码子的替代物。我们想知道 移码被激发了首先，我们将确定有多少个7 nt +1 通过随机寡核苷酸诱变存在移码位点。第二、 翻译GCG的tRNA似乎具有特殊的能力， 移码而不会在mRNA模板上滑动。我们将 试图确定这个和其他“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也可能是驱动移码到+1框的特殊密码子; 我们将通过过量表达和诱变tRNA来检验这一假设。 Ty 3移码是由下游的“上下文”刺激的，尽管我们没有 知道怎么做我们将要检验的一些假设是， 上下文的产物扰乱了框架转换，或者上下文， 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 寻找特定的A和P位点tRNA与其他 翻译机制的组成部分，以确定反式作用因子 对框架转换至关重要。 这些研究将为理解 程序化的移码位点与翻译的 机械.这些研究的结果将与我们的 了解核糖体作为一个分子机器， 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.