Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae.
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.[1]References
- Construction and analysis of deletions in the amino-terminal extension of glutamine tRNA synthetase of Saccharomyces cerevisiae. Ludmerer, S.W., Schimmel, P. J. Biol. Chem. (1987) [Pubmed]
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