lacZ translation initiation mutations.
lacZ translation initiation mutations.
复制标题
lacZ 翻译起始突变。
DOI：
10.1016/0022-2836(84)90043-3
复制
发表时间：
1984
影响因子：
5.6
通讯作者：
Reznikoff,WS
中科院分区：
文献类型：
--
作者：
Munson,LM;Stormo,GD;Niece,RL;Reznikoff,WS
目前已分离到16个接近lacZ基因起始点的单点突变，并测定了它们对lacZ表达的影响。获得了五个突变，它们改变了信使RNA中掩盖起始密码子的潜在的茎和环结构。这种茎和环的形成是在克隆调控区域过程中引入的DNA序列转录的结果。分离出的突变随后被转移到删除这种结构的背景中。对这些突变的分析表明，二级结构抑制了lacZ表达5.8倍，单点突变或9个碱基对缺失都可以完全解除这种抑制。此外，研究还发现，起始密码子之后第一个碱基的A到C的颠倒(在没有抑制性二级结构的情况下)使Z的表达减少了近两倍，而接下来两个位置的C到U的转换几乎没有影响。在存在或不存在涉及两个起始密码子的二级结构的情况下，研究了这些突变的影响。结果发现，当翻译起始被二级结构抑制时，增加Shine-Dalgarno序列的长度使LacZ表达增加2.8倍，减少该序列的长度使LacZ表达减少12倍。当翻译起始不被二级结构抑制时，增加Shine-Dalgarno序列的长度没有影响，减少该序列的长度只会使lacZ表达减少6倍。两个起始密码子位于LacZ基因的开头，分别位于Shine-Dalgarno序列的7个和13个碱基。NH2末端序列分析表明，大多数合成的蛋白质起始于野生型acZ基因的第一个起始密码子(与J.L.Brown和他的同事以前报道的结果一致)。当引入导致mRNA二级结构改变的序列时，两个起始密码子的使用量几乎相等。获得了位于第一起始密码子的3个突变株和2个假回复株。研究发现，当第一个起始密码子从Aug改为Gug时，该密码子的翻译起始量减少了10倍。第一起始密码子上的其他突变导致起始仅发生在第二起始密码子上，其效率由位于间隔区的核苷酸决定。
Sixteen single point mutations near the beginning of thelacZgene have been isolated and their effect onlacZexpression has been measured. Five mutations were obtained that alter a potential stem-and-loop structure in the messenger RNA that masks the initiation codons. Formation of this stem-and-loop is a result of transcription of DNA sequences introduced during the cloning of thelacregulatory region. The mutations isolated were then moved into a background that deleted this structure. Analysis of these mutations indicated that the secondary structure inhibitedlacZexpression 5.8-fold and that either single point mutations or a 9 base-pair deletion could relieve this inhibition completely. In addition, it was found that an A to C transversion in the first base following the initiation codon (in the absence of the inhibitory secondary structure) decreaseslacZexpression almost twofold, whereas C to U transitions in the next two positions have negligible effects.Mutations were also obtained that either increase or decrease the length of the Shine-Dalgarno sequence. The effects of these mutations were studied in the presence or absence of the secondary structure that involves the two initiation codons. It was found that when translation initiation was inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence increasedlacZexpression 2.8-fold and decreasing the length of this sequence reducedlacZexpression 12-fold. When translation initiation was not inhibited by the secondary structure, increasing the length of the Shine-Dalgarno sequence had no effect and decreasing the length of this sequence only reducedlacZexpression sixfold. The mechanistic implications of these results are discussed.Two initiation codons are located in the beginning of thelacZgene, 7 and 13 bases from the Shine-Dalgarno sequence. NH2-terminal sequence analysis indicated that the majority of the protein synthesized initiate at the first initiation codon in the wild-typelacZgene (in agreement with results reported previously by J. L. Brown and his colleagues). Upon introduction of sequences that result in a change in the mRNA secondary structure, both initiation codons are used in almost equal amounts. Three mutations and two pseudorevertants were obtained, which are located in the first initiation codon. It was found that when the first initiation codon is changed from AUG to GUG, translation initiation is decreased tenfold at that codon. Other mutations at the first initiation codon resulted in initiation occurring exclusively at the second initiation codon at an efficiency that is determined by the nucleotides located in the spacer region.