SITE-SPECIFIC TRANSLATIONAL FRAMESHIFTING

特定地点的平移框架转移

• 批准号: 3277093
• 负责人: Philip James Farabaugh
• 金额: $ 13.16万
• 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 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277093
• 关键词: RNA splicing; Saccharomyces; endonuclease; frameshift mutation; fungal genetics; gel electrophoresis; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic promoter element; genetic recombination; genetic transcription; genetic translation; molecular cloning; molecular genetics; nucleic acid hybridization; nucleic acid sequence; oligonucleotides; site directed mutagenesis; transposon /insertion element

The concept of genetic colinearity between the DNA and protein sequences has been found to be violated in many genes during the last ten years. The most obvious example is RNA splicing. Many other examples exist involving unusual mechanisms of transcription and translation. Each of these unusual modes of expression provide molecular biology with new tools to study fundamental genetic processes. We are studying an unusual very efficient ribosomal frameshift which occurs in the Ty elements of the yeast Saccharomyces cerevisiae. The Ty elements of yeast are a dispersed family of 30 related retroviral-like transposons. The elements include two genes, TYA and TYB, which are analogous to gag and pol of retroviruses. TYB is expressed as a fusion to the TYA gene. This fusion could occur by splicing or RNa editing. However since the RNA sequence of the overlap between the two genes is colinear with the DNA only translational models explain TYB expression. We refer to them generically as frameshifting, realizing that mechanism has not been defined. A 14 nucleotide sequence is both necessary and sufficient to promote 20% frameshifting; and that it is the site of the frameshift. This sequence includes no obvious secondary structure, but does include an unusual codon, AGG. This codon is recognized by a rare tRNA encoded by a single genetic locus. It may be that a translational pause induced by the low abundance of the tRNA is required for the frameshift to occur. Many questions remain unanswered about Ty site-specific translational frameshifting. What nucleotides are essential to the process? Would overproduction of the cognate tRNA eliminate frameshifting dependent upon the AGG codon? Could other codons recognized by rare tRNAs substitute? What external factors (ribosomal proteins? translation factors?) regulate the event? Is translational termination required immediately distal to the frameshift site? Frameshifting promoted by the 14 nucleotide minimal site is suppressed near the TYA initiator AUG. This suppression may be caused by the context surrounding the site, though this seems unlikely. Alternatively the early steps in elongation could be different in some way which precludes frameshifting. Frameshifting may operate in a frame-independent way, though most efficient when occurring between the TYA and TYB frames. This result is difficult to reconcile with a model which requires a specific codon-anticodon interaction at the frameshift site. Biochemical analysis can best demonstrate the site of the Ty frameshift. We are purifying the product of a chimeric gene in which a portion of the S. aureus protein A gene is expressed dependent upon the 14 nucleotide minimal site. The sequence of a cyanogen bromide fragment encompassing the frameshift site and protein A fragment will be determined on a gas-phase sequenator. We are also using in vitro translation to demonstrate that an RNA colinear with the DNA can allow TYB expression.

DNA与蛋白质的遗传共线性概念 已经发现在许多基因中， 过去十年。 最明显的例子是RNA剪接。 许多 还有一些例子涉及到不寻常的转录机制 和翻译。 这些不寻常的表达方式中的每一种都提供了 分子生物学与研究基础遗传学的新工具 流程. 我们正在研究一种不寻常的高效核糖体 发生在酵母的Ty元件中的移码 酿酒酵母 酵母的Ty元件是30个相关的分散的家族 逆转录病毒样转座子。 这些元件包括两个基因，TYA 和TYB，它们类似于逆转录病毒的gag和pol。 TYB 表达为TYA基因的融合体。 这种融合可能发生在 通过剪接或RNa编辑。 然而，由于RNA序列的 两个基因之间的重叠仅与DNA共线， 翻译模型解释TYB表达。 我们称之为 一般来说是框架转换，意识到机制还没有被 定义了 14个核苷酸的序列是必要的，也是足够的， 促进20%的移码;它是网站的 移码 该序列不包括明显的二级结构， 但包含一个不寻常的密码子AGG 这个密码子被识别 一种由单一基因位点编码的罕见tRNA 可能是 由低丰度的tRNA诱导的翻译暂停， 进行移码所需的信息 还有许多问题 Ty位点特异性翻译移码的问题 什么样的核苷酸对这个过程至关重要？ 将 同源tRNA的过量产生消除了移码 依赖于AGG密码子 其他密码子是否能被 罕见的tRNA替代品？ 什么外部因素（核糖体蛋白？ 翻译因素？）规范事件？ 是平移 终止需要立即远端移码网站？ 由14个核苷酸的最小位点促进的移码是 在TYA引发剂AUC附近受到抑制。这种抑制可能是 这是由该网站周围的环境造成的，尽管这似乎是 不太可能 或者，伸长的早期步骤可以是 在某种程度上是不同的，这就排除了框架转换。 移码 可以以独立于帧的方式操作，尽管当 发生在TYA和TYB帧之间。 这个结果是困难的 与需要特定密码子-反密码子的模型相一致 在移码位点的相互作用。 生化分析可以最好地显示Ty的位置 移码 我们正在纯化嵌合基因的产物， 这是S的一部分。金黄色葡萄球菌蛋白A基因表达 这取决于14个核苷酸的最小位点。 的序列 包含移码位点的溴化氰片段， 蛋白A片段将在气相测序仪上测定。 我们还使用体外翻译来证明RNA 与DNA共线可以允许TYB表达。