Construction of an Escherichia coli knockout strain for functional analysis of tRNAAsp

The specific aminoacylation of tRNA is critical for translation of the genetic code. A molecular description of aminoacylation requires knowledge of the relevant three-dimensional structures, biochemical parameters and the structure-function relationship of the synthetase and its substrate tRNA. Extensive structural and biochemical data are available on the aspartic acid system of Escherichia coli, but there is a paucity of cellular functional data. We have developed a system to overcome this deficiency by engineering an E. coli knockout tRNA(Asp) strain, thereby allowing a penetrating analysis of tRNA(Asp) structure and function under conditions that prevail in the cell. (C) 2001 Academic Press.