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[52] Assay for AA-tRNA recognition by the EFTu-GTP complex of Escherichia coli

1974; Academic Press; Linguagem: Inglês

10.1016/0076-6879(74)29057-8

1557-7988

Tames Ofengand,

Bacterial Genetics and Biotechnology

Recognition of AA-tRNA (but not tRNA) by the EFTu-GTP complex of Escherichia coli and by the EFI-GTP complex of other organisms is central to the process of protein synthesis as this complex is an essential intermediate in the process by which all AA-tRNA's become attached to their proper site on the ribosome. The prerequisite formation of this intermediate also provides a way to screen out unwanted tRNA's such as deacylated tRNA or N-acylated AA-tRNA that might otherwise block the protein-synthesizing machinery. A number of modifications of AA-tRNA's have been studied in an attempt to map out the regions of the tRNA that are essential to interaction with the Tu-GTP complex. This chapter describes a simple technique for measuring this interaction, based on the observations that while EFTu and EFTu-GTP complexes adsorb to nitrocellulose membrane filters, the ternary complex of AA-tRNA-EFTu-GTP does not. This effect is the basis of the present assay. The chapter also describes a second method for use when it is not certain that the modified AA-tRNA-EFTu-GTP ternary complex, if formed, would pass through the filter. In this method, the test AA-tRNA competes with a standard AA-tRNA for a limiting amount of EFTu-GTP.