Mutants affecting tRNA^Phe from Escherichia coli Studies of the suppression of thermosensitive phenylalanyl‐tRNA synthetase

Abstract

Four mutants of phe V, a gene coding for tRNAPhe 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 tRNAPh', is expressed about 10-fold lower in transformed cells than wild-type phel': 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 tRNAPh'.A gene, p h e v coding for tRNAPhe in Escherichia coli, was isolated in this laboratory by virtue of the fact that plasmids or cosmids carrying pheV could complement the thermosensitivity of certain mutants affecting phenylalanyl-tRNA synthetase (PheRS) [l]. Subsequently, mutants in pheV were obtained after mutagenesis of the gene in vitro [2]. Two criteria were employed in screening for mutants of pheV: (a) deattenuation of b-galactosidase synthesis, while under the control of the attenuator region of the pheS, T operon (which codes for PheRS) by means of an operon fusion: (b) loss of the ability to complement the thermosensitivity of a mutant form of PheRS. By this approach mutants affecting four different positions in tRNAPhe were isolated [2]. All four mutants lose the ability of the wild-type tRNA to complement thermosensitive PheRS. Although the mechanism for the complementation by plasmids carrying wild-type phe V is not entirely understood, it probably involves an increase in the activity or stability of the mutant PheRS in the presence of an excess of tRNAphe, maybe associated with an increased K , for tRNA of the mutant enzyme. Loss of complementation by mutations affecting the structural gene for tRNAPhe may thus be due to less efficient transcription or maturation or an altered interaction with PheRS.Here we describe experiments which investigate the mechanism of loss of complementation by the mutant species of tRNAPhe. Furthermore, we show that one of these mutants may be used to isolate promoter mutants in pheV with increased promoter activity. MATERIALS AND METHODS Strains and plasmidsStrains and plasmids have been described in [3], except for plasmids pPPl5, a tetracycline-resistant derivative of pBR322 with a 350-base-pair insertion which includes a gene for tRNAPhe (phev), [4] and pUC19 [5]. Strains IBPC 5311 or IBPC 1671 transformed by plasmi...

The growth defect in Escherichia coli deficient in peptidyl-tRNA hydrolase is due to starvation for Lys-tRNA(Lys).

“…tRNA amino acid acceptor capacity tRNA was extracted with 70% phenol (Cox and Littauer, 1962) from cells grown to an optical density of 1.2 at 600 nm in LB medium and washed once with 0.9% NaCl. Amino acid acceptance was determined as described by Delamarche et al (1987). Amino acid acceptance was determined as described by Delamarche et al (1987).…”

“…By cloning a similar region from the non-suppressor strain Y1, suppression was shown not to depend on any mutation within the cloned region, a conclusion confirmed by using plasmid pOF12 (Fayet et al, 1986), carrying a wild-type groESL region on an 8 kb EcoRI fragment. Futhermore, co-transduction experiments using a kanamycin-resistant cassette inserted immediately downstream of the groEL gene (Zeilstra-Ryalls et al, 1993) as selected marker indicated that the 94 min chromosomal suppressor muta- Delamarche et al (1987) and Caillet et al (1985) tRNAPro3 proM ptRNAHIsCCA O'Connor et al (1993) tRNASerS serW pTH211 Cummings et al (1994) tRNAThrr thrW pTH2 Komine and Inokuchi (1990) tRNAThr3 thrT pBSBA2 Springer et al (1989) tRNAThr4 thrU pBSARl Springer et al (1989) tRNAVal valU pVH94 this work…”

“…After ethanol precipitation, the tRNA was purified by chromatography on DE52 (Whatmann) and deacylated by incubation for 30 min at 37°C in 1 M Tris-HCI, pH 9. Amino acid acceptance was determined as described by Delamarche et al (1987).…”

Common sequence determinants of the response of a prokaryotic promoter to DNA bending and supercoiling.

“…It appears, therefore, that the step affected by the -7 mutation is rate-limiting only under certain promoter weakening conditions. Possibly, this is a general property of mutants in this region (Delamarche et al, 1987). Suppression of the -70 lesion suggests that the need for upstream activation is bypassed upon removing the supercoiling-sensitive step (see Discussion).…”

Identification of the pheS5 mutation, which causes thermosensitivity of Escherichia coli mutant NP37

“…(ii) The PheRS genes from Bacillus subtilis (5) and both genes for tRNAPhe (28), pheV(6, 7) and pheU (12, 33), were cloned by complementation and phenotypic reversal of the thermosensitivity caused bypheS5. (iii) Various mutant pheS (23) and tRNAPhe (10,38) gene products were functionally analyzed in pheS5 backgrounds, and overproduction of partially undermodified tRNAPhe in strain NP37 was used to study the impact of posttranscriptional tRNA modification on aminoacylation and codon recognition (39). (iv) pheS5 strains were involved in the discovery of attenuation mechanisms in the control of the pheA gene and pheST operon expression (4,26,29,36,37).…”