Ludmerer S W, Schimmel P
J Biol Chem. 1987 Aug 5;262(22):10807-13.
GLN4 of Saccharomyces cerevisiae encodes an amino-terminal extension of 224 amino acids. This is connected to a polypeptide which is colinear with and 40% identical to Escherichia coli glutamine tRNA synthetase. We examined the potential significance of the amino-terminal extension. Two single base and five multiple base frame shift deletions were constructed in this segment. Each of these mutations is associated with a lethal phenotype. This suggests that the coding sequence for the amino-terminal extension is translated. It also implies that there are no translation restarts downstream of the coding region for the amino-terminal extension which produce active enzyme. Three internal deletions of various sizes, and which preserve the correct reading frame, were constructed in the coding region of the amino-terminal extension. Cells which harbor such in-frame deletions on a multi copy plasmid are viable, even when a deletion construct is the only source of GLN4-encoded activity. Extracts of cells which have one of these deletions have reduced, but measurable, glutamine tRNA synthetase activity. We conclude that the catalytic activity resides with the segment which is homologous to the E. coli enzyme and that the amino-terminal extension itself is dispensable for aminoacylation activity. Each of the internal in-frame deletion constructions is respiration-proficient. The amino-terminal extension, therefore, is not used for an essential mitochondrial function of the GLN4 gene product. Within the accuracy of the measurements, activities of four other aminoacyl-tRNA synthetases are not affected by the presence of a GLN4 internal deletion allele as the only source of GLN4-encoded activity. This suggests that the amino-terminal extension does not stabilize a complex which includes one or more of these four enzymes and whose activity depends on proper assembly of the complex.
酿酒酵母的GLN4编码一个由224个氨基酸组成的氨基末端延伸序列。它与一种多肽相连，该多肽与大肠杆菌谷氨酰胺tRNA合成酶共线性且有40%的同源性。我们研究了氨基末端延伸序列的潜在意义。在该片段中构建了两个单碱基和五个多碱基移码缺失突变。这些突变中的每一个都与致死表型相关。这表明氨基末端延伸序列的编码序列被翻译了。这也意味着在氨基末端延伸序列的编码区域下游不存在产生活性酶的翻译重新起始现象。在氨基末端延伸序列的编码区域构建了三个不同大小且保持正确阅读框的内部缺失突变。即使缺失构建体是GLN4编码活性的唯一来源，携带多拷贝质粒上此类框内缺失突变的细胞仍然是存活的。具有这些缺失突变之一的细胞提取物中谷氨酰胺tRNA合成酶活性降低，但仍可检测到。我们得出结论，催化活性存在于与大肠杆菌酶同源的片段中，并且氨基末端延伸序列本身对于氨酰化活性是可有可无的。每个内部框内缺失构建体的细胞呼吸功能正常。因此，氨基末端延伸序列不用于GLN4基因产物的必需线粒体功能。在测量精度范围内，当GLN4内部缺失等位基因作为GLN4编码活性的唯一来源时，其他四种氨酰-tRNA合成酶的活性不受影响。这表明氨基末端延伸序列不会稳定一个包含这四种酶中的一种或多种且其活性依赖于复合物正确组装的复合物。
