Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem.
Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem.
复制标题
酵母脯氨酸 tRNA 的剪接，其中反密码子茎中含有新的抑制突变。
DOI：
10.1016/0022-2836(86)90463-8
复制
发表时间：
1986
影响因子：
5.6
通讯作者：
Knapp,G
中科院分区：
文献类型：
--
作者：
Winey,M;Mendenhall,MD;Cummins,CM;Culbertson,MR;Knapp,G
酿酒酵母中含有脯氨酸内含子的tRNAUGG基因可以通过39位G到U碱基替换突变来抑制脯氨酸密码子中的+1移码突变。该突变改变了3 ′剪接点，破坏了反密码子茎的底部碱基对，这可能使tRNA能够读取四碱基密码子。为了了解抑制机制并研究抑制性前体tRNA的剪接，我们在体内测定了成熟野生型和突变型抑制基因产物的序列，并在体外分析了相应前体tRNA的剪接。我们发现，一种新的tRNA抑制菌株分离的移码抑制基因的产物。序列分析表明，抑制基因前tRNA在与野生型前tRNA相同的位点剪接。因此，tRNA包含一个四个碱基的反密码子茎和九个碱基的反密码子环。体外抑制性前tRNA分析显示，与野生型相比，3 ′剪接点处的核酸内切酶切割效率降低。此外，减少积累的成熟抑制tRNA的结合切割和连接反应中观察到。这些结果表明，在3 ′剪接点的切割是无效的，但没有消除。来自抑制菌株的新型tRNA被证明是通过删除抑制基因的内含子而起抑制作用的功能剂。该基因在体内产生的tRNA与内含子+基因产物的tRNA相同，表明内含子不需要进行适当的碱基修饰。内含子−基因的产物是比内含子+基因的产物更有效的抑制子。对这一结果的一种解释是，体内低效率的剪接可能限制了成熟抑制性tRNA的稳态水平。
The intron-containing proline tRNAUGGgenes inSaccharomyces cerevisiaecan mutate to suppress +1 frameshift mutations in proline codonsviaa G to U base substitution mutation at position 39. The mutation alters the 3′ splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene productsin vivoand analyzed splicing of the corresponding pre-tRNAsin vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNAin vitrorevealed that endonuclease cleavage at the 3′ splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3′ splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA producedin vivofrom this gene is identical to that of the product of an intron+gene, indicating that the intron is not required for proper base modification. The product of the intron−gene is a more efficient suppressor than the product of an intron+gene. One interpretation of this result is that inefficient splicingin vivomay be limiting the steady-state level of mature suppressor tRNA.