OVERPRODUCTION AND PURIFICATION OF NATIVE AND QUEUINE-LACKING ESCHERICHIA-COLI TRANSFER RNA(ASP) - ROLE OF THE WOBBLE BASE IN TRANSFER RNA(ASP) ACYLATION

Escherichia coli tRNAAsp was overproduced in E. coli up to 15-fold from a synthetic tRNAAsp gene placed in a plasmid under the dependence of an isopropyl-β, D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNAAsp[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNAAsp, the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNAAsp. Kinetic properties of tRNAAsp[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNAAsp and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNAAsp. Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34. © 1993 Academic Press Limited.