Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.
Mutations in the anticodon stem affect removal of introns from pre-tRNA in Saccharomyces cerevisiae.
复制标题
反密码子茎的突变影响酿酒酵母前 tRNA 内含子的去除。
DOI：
10.1128/mcb.9.10.4220-4228.1989
复制
发表时间：
1989
影响因子：
5.3
通讯作者：
Knapp,G
中科院分区：
文献类型：
--
作者：
Mathison,L;Winey,M;Soref,C;Culbertson,MR;Knapp,G
为了评价外显子结构域在tRNA剪接中的作用，通过suf8基因的定点诱变改变了来自酿酒酵母的脯氨酸前tRNAUGG的反密码子干。构建了 16 个等位基因，编码突变体前 tRNA，其中包含与反密码子环相邻的反密码子茎的最后一个碱基对（位置 31 和 39）中的所有可能的碱基组合。通过体外核酸内切试验筛选改变的前 tRNA，以确定二级结构的扰动是否影响内含子切除反应。只要通过标准碱基配对或 G U 相互作用维持反密码子茎中的二级结构，前 tRNA 就会被有效切割。然而，大多数二级结构被破坏的前 tRNA 是内含子切除的不良底物。我们还确定了suf8等位基因在体内产生功能性产物的程度。每个等位基因以一到三个拷贝整合到酵母染色体中，或通过转化引入高拷贝数质粒上。功能产物的形成通过每个等位基因通过四碱基密码子读取抑制+1移码突变his4-713的能力来测定，如先前针对SUF8-1抑制等位基因所示。我们发现，在反密码子茎中位置 31/39 处包含任何标准碱基对或 GU 对的等位基因未能抑制his4-713。我们无法评估这些等位基因的体内剪接，因为 tRNA 产品，即使它们被制成，也预计会读取正常的三联体而不是四联体密码子。然而，所有在反密码子茎中第 31/ 39 位含有破坏碱基对的等位基因都以引起移码抑制的方式改变了 tRNA 的结构。抑制表明剪接一定在体内发生了某种程度，尽管大多数抑制等位基因产生的前tRNA在体外切割效率低或根本不切割。这些结果对于一般体外切割测定的解释以及潜在使用抑制器来选择影响 tRNA 剪接的突变具有重要意义。
To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGGfromSaccharomyces cerevisiaewas altered by site-directed mutagenesis of thesuf8gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which thesuf8alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutationhis4-713through four-base codon reading, as shown previously for theSUF8-1suppressor allele. We found that alleles containing any standard base pair or G U pair at position 31/39 in the anticodon stem failed to suppresshis4-713.We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppressor alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affect tRNA splicing.