SITE-SPECIFIC TRANSLATIONAL FRAMESHIFTING

DNA与蛋白质遗传共线性的概念 序列已被发现在许多基因中被破坏 过去的十年里。最明显的例子是RNA剪接。许多 还有一些例子涉及不寻常的转录机制。 还有翻译。这些不寻常的表达方式中的每一种都提供了 分子生物学与基础遗传学研究的新工具 流程。我们正在研究一种不同寻常的非常有效的核糖体 发生在酵母的Ty元素中的移码现象 酿酒酵母。 酵母中的Ty元素是一个分散的家族，有30个相关的 类似逆转录病毒的转座子。这些元素包括两个基因，TYA 和TYB，类似于逆转录病毒的Gag和Pol。类型 表达为与TYA基因的融合。这种聚变可能会发生 通过剪接或RNA编辑。然而，由于该病毒的RNA序列 这两个基因之间的重叠只与DNA共线 翻译模型解释了TYB的表达。我们指的是它们 一般称为帧移动，意识到机制尚未 已定义。 14个核苷酸序列既是必要的，也是充分的 促进20%的帧移位；它是 移帧。该序列没有明显的二级结构， 但它确实包含了一个不寻常的密码子agg。该密码子被识别 由单一遗传位点编码的一种罕见的tRNA。可能是因为 TRNA丰度低引起的翻译暂停是 发生移码所必需的。许多问题仍然存在 关于Ty站点特定的翻译框架转换的问题未得到回答。 在这个过程中，哪些核苷酸是必不可少的？会 同源tRNA的过量生产消除了移码 依赖AGG密码子吗？其他密码子是否能被 稀有的tRNA替代品？哪些外部因素(核糖体蛋白？ 翻译因素？)监管这一事件？具有翻译性 是否需要立即终止移码站点的远端？ 由14个核苷酸最小位点促进的移码 在TYA发起者附近被抑制。这种压制可能是 由网站周围的环境造成的，尽管这看起来 不太可能。或者，伸长的早期步骤可以是 在某种程度上不同，这排除了移帧。帧移位 可以以独立于帧的方式运行，但在以下情况下效率最高 发生在TYA和TYB帧之间。这个结果很难实现 与需要特定密码子-反密码子的模型进行协调 移码位置的相互作用。 生化分析能最好地显示TY的位置 移帧。我们正在提纯嵌合基因的产物 金黄色葡萄球菌蛋白A基因的哪一部分被表达 依赖于14个核苷酸的最小位点。的序列 包含移码位置的溴化氰片段和 蛋白A片段将在气相测序仪上测定。 我们还在使用体外翻译来证明一种RNA 与DNA共线可允许TYB表达。

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.