Mutants affecting tRNA(Phe) from Escherichia coli. Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase.
Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase. One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV. This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase. The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).
Delamarche
C
C
Institut de Biologie Physico-Chimique, Paris, France.
Vacher
J
J
Buckingham
R H
RH
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005786:Gene Expression Regulation; D005796:Genes; D066298:In Vitro Techniques; D010652:Phenylalanine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics*; D012360:RNA, Transfer, Phe / Q000235:genetics*; D013489:Suppression, Genetic; D013696:Temperature
D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D000604:Amino Acyl-tRNA Synthetases; D010652:Phenylalanine-tRNA Ligase