SLIPPERY RUNS, SHIFTY STOPS, BACKWARD STEPS, AND FORWARD HOPS - -2, -1, +1, +2, +5, AND +6 RIBOSOMAL FRAMESHIFTING
SLIPPERY RUNS, SHIFTY STOPS, BACKWARD STEPS, AND FORWARD HOPS - -2, -1, +1, +2, +5, AND +6 RIBOSOMAL FRAMESHIFTING
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DOI：
10.1101/sqb.1987.052.01.078
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发表时间：
1987-01-01
期刊：
影响因子：
--
通讯作者：
GESTELAND, RF
中科院分区：
文献类型：
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作者：
WEISS, RB;DUNN, DM;GESTELAND, RF
移码突变经常表达残留水平的基因活性；也就是说，它们经常是漏水的。这种泄漏可以作为一种工具来定义在基因表达过程中影响阅读框的功能成分（Atkins et al. 1972; Fox and Weiss- brummer 1980; Weiss and Gallant 1983）。最近在有效构建合成DNA序列的能力方面的技术进步促进了小的，定义的“移码窗口”的构建。这些窗口是可以检测和测量移码事件的区域。克隆的合成窗口被融合到一个活性半乳糖苷酶基因的5'编码区，该基因为移码事件提供了一个敏感的监测。融合到lacZ基因上的优点是对半乳糖苷酶活性进行简单的比色测定，并且融合序列对酶的特定活性或稳定性的影响很小或没有影响（Miller和Albertini 1983）。移码事件也在从窗口的mRNA序列翻译的蛋白质序列中留下了其特征的线索。含有移码的/3-半乳糖苷酶的产量和纯度足以确定其氨基端序列，这为移码的发生提供了确凿的证据，该序列可用于推断产生读框丢失的事件类型。移码窗口在它们的3‘边界处由相对于翻译开始的0帧中的停止密码子定义，在它们的5’边界处由监视的输出帧中的停止密码子定义。核糖体只能通过在零帧翻译进入窗口，它们只能通过移动到-1或+ 1帧退出窗口，其中只有一个导致活性/3-半乳糖苷酶的产生。监视1、0或+ 1帧的窗口被指定为2p、3p或4p。移码窗口还具有确定移码发生的水平（复制或转录与翻译）的形式属性。这种特性包括“重构”窗口的序列，使序列保持不变，但核糖体通过它翻译的框架都被移动了。由于核糖体读取读框，翻译框移应该对这种重构敏感，但由于参与这些过程的酶机制尚不知道如何检测读框，因此转录移位或移码的遗传逆转水平不应因重构而改变。
Frameshift mutations frequently express residual levels of gene activity; that is, they are often leaky. This leakiness can be used as a tool to define the functional components that affect the reading frame during gene expression (Atkins et al. 1972; Fox and Weiss-Brummer 1980; Weiss and Gallant 1983). Recent technological advances in the capability to efficiently build synthetic DNA sequences have facilitated the construction of small, defined" frameshift windows." These windows are regions where frameshift events can be detected and measured. The cloned synthetic window is fused onto the 5'coding region of an active fl-galactosidase gene that provides a sensitive monitor for the frameshift events. Fusions onto the lacZ gene have the advantages of simple colorimetric assays for fi-galactosidase activity and little or no effect of the fused sequence on the specific activity or stability of the enzyme (Miller and Albertini 1983). A frameshift event also leaves a clue to its character in the protein sequence translated from the window's mRNA sequence. Recovery of the frameshift-containing/3-galactosidase in sufficient yield and purity for determining its amino-terminal sequence provides hard evidence for the occurrence of a frameshift, and this sequence may be used to infer the kind of event generating the loss of reading frame.Frameshift windows are defined at their 3'border by a stop codon in the zero frame relative to the translation start and at their 5'border by a stop codon in the monitored outgoing frame. Ribosomes can only enter the window by translating in the zero frame, and they can only exit by shifting to the-1 or+ 1 frame, only one of which results in the production of active/3-galactosidase. A 2p, 3p, or 4p designation is given to windows that monitor the-1, 0, or+ 1 frame. Frameshift windows also have as a formal property the ability to determine the level (replication or transcription versus translation) at which the shift occurs. This property involves" reframing" the window's sequence so that the sequence remains the same but the frames in which the ribosome translates through it have both been shifted. Translational frameshifts should be sensitive to such reframing since ribosomes read frame, but the levels of transcriptional shifts or genetic reversion of the frameshift should not be altered by reframing, since the enzymatic machinery involved in these processes is not known to detect the reading frame.