Overproduction and purification of native and queuine-lacking Escherichia coli tRNA(Asp). Role of the wobble base in tRNA(Asp) acylation.
Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[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 tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[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 tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). 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.
Martin
F
F
Institut de Biologie Moléculaire et Cellulaire du Centre National de la Recherche Scientifique, Strasbourg, France.
Eriani
G
G
Eiler
S
S
Moras
D
D
Dirheimer
G
G
Gangloff
J
J
D000926:Anticodon; D001226:Aspartate-tRNA Ligase / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013329:Structure-Activity Relationship; D046249:Transfer RNA Aminoacylation
D000926:Anticodon; D012349:RNA, Transfer, Asp; D006147:Guanine; C019549:queuine; D001226:Aspartate-tRNA Ligase