Identiflcation of clones an<i>E.coli</i>tRNA<sup>phe</sup>gene by suppression of pheylananyl-tRNA synthetase thermosensitive mutants

Caillet, Joeèl; Plumbridge, Jacqueline; Springer, Mathias; Vacher, Jean; Delamarche, Christian; Buckingham, Richard H.; Grunberg‐Manago, Marianne

doi:10.1093/nar/11.3.727

Cited by 19 publications

(9 citation statements)

References 14 publications

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“…Previously reported increases in aminoacylation for monomeric or dimeric E. coli tRNA genes cloned into E. coli are around two-to ninefold (3,8,20). The tRNA genes close to the promoter on the B. subtilis tRNA gene segment fell into this range; however, the levels of aminoacylation decreased for tRNA species from the more distal tRNA genes.…”

Section: Discussionmentioning

confidence: 86%

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Vold¹,

Green²

1986

J Bacteriol

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A cloned DNA segment from Bacillus subtilis containing 21 tRNA genes was introduced into Escherichia cQli. In the B. subtilis genome, these tRNA genes are locatted after an rRNA gene set and before tandem terminators.The rRNA and tRNA genes are thought to represent a single transcriptiqnal unit. However, another putative promoter occurs after the second tRNA gene within the tRNA gene cluster and has a sequence compatible with both the major B. subtilis (sigma 43 type) promoter and the major E. coli promoter. The B. subtilis 21-tRNA-gene cluster was introduced into E. coli to see whether this promoter would be recognized in E. coli, to determine the start point of transcription in the E. coli system, and to see whether mature B. subtilis tRNAs would be transcribed and processed in E. coli. Expression was evaluated by monitoring levels of aminoacylation of mature tRNAs extracted from E. coli containing plasmids with or without the B. subtilis tRNA genes and by examining profiles of isoaccepting species on columns of RPC-5. Si nuclease mapping was performed to define the starting point for transcription. The results indicated that a putative promoter located within the R. subtills tRNA gene region was functional when cloned into E. coli and that it initiated at the same nucleotide as it does in B. subtilis. In addition, at least some B. subiilis tRNA genes could be transcribed and processed in E. coli to mature tRNAs capable of accepting an amino acid.

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Section: Discussionmentioning

confidence: 86%

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Vold¹,

Green²

1986

J Bacteriol

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“…For example, it is conceivable that clones carrying B. subtilis tRNAPhe could complement, since thermosensitive E. coli strains carrying pheS or pheT are complemented by the cloned E. coli tRNAPhe gene (3).…”

Section: 611mentioning

confidence: 99%

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis

et al. 1989

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The genes that encode the two subunits of Bacillus subtilis phenylalanyl-tRNA synthetase were cloned from a X library of chromosomal B. subtilis DNA by specific complementation of a thermosensitive Escherichia coli pheS mutation. Both genes (we named them pheS and pheT, analogous to the corresponding genes of E. colt) are carried by a 6.6-kilobase-pair PstI fragment which also complements E. coli pheT mutations. This fragment directs the synthesis of two proteins identical in size to the purified a and 1 subunits of the phenylalanyl-tRNA synthetase of B. subtilis with Mrs of 42,000 and 97,000, respectively. A recombinant shuttle plasmid carrying the genes caused 10-fold overproduction of functional phenylalanyl-tRNA synthetase in B. subtilis.Aminoacyl-tRNA synthetases play an integral role in protein biosynthesis by catalyzing the attachment of amino acids to the 3' ends of their cognate tRNAs (8). Furthermore, aminoacyl-tRNA synthetases are directly or indirectly involved in several other cellular processes (1,8).In Escherichia coli, 11 genes that encode these enzymes have been cloned, and their regulation has been studied at various levels (8). In Bacillus subtilis, however, very little is known about aminoacyl-tRNA synthetases, there are no reports of genes being cloned, and only two genes have been mapped, those for the tryptophanyl-tRNA (33) and lysyltRNA (25) synthetases. Here we report the cloning of the genes, for B. subtilis phenylalanyl-tRNA synthetase; this is the first report of a synthetase cloned from the gram-positive bacterium B. subtilis.The phenylalanyl-tRNA synthetase of B. subtilis is competitively inhibited by the phenylalanine analog ochratoxin A, a mycotoxin produced and excreted by several species of the genera Aspergillus and Penicillium. It contaminates food and feeds on and acts in animals as a nephrotoxin. With B. subtilis, an inducible resistance to ochratoxin A has been observed in vivo, which was interpreted as being due to an increase in the amount of phenylalanyl-tRNA synthetase in induced cells (26). The cloning of the phenylalanyl-tRNA synthetase genes of B. subtilis is a first step in an investigation of this regulatory phenomenon.When we started this work, the phenylalanyl-tRNA synthetase of B. subtilis was not purified and so the methods of cloning the genes by using antibodies to the enzyme could not be used. Since the E. coli genes pheST had been cloned (11) and sequenced (19) thermosensitive E. coli phenylalanyl-tRNA synthetase in vivo.The bacterial strains, bacteriophages, and plasmids used in this study are listed in Table 1. A genomic X library of B. subtilis was constructed by cloning 9 to 20 kilobase pairs (kbp) of partial Sau3A1 digestion fragments of chromosomal DNA from wild-type B. subtilis 168 into X L47.1 digested with BamHI. The ligation mixture was packaged in vitro by using the packaging lysates of an E. coli C strain (SMR10) lacking the K restriction system (27,28). Packaged phages are amplified in the P2 lysogen E. coli Q359 to select for Spirecombinants....

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“…As a result, such studies have rarely been pursued to the point of detailed kinetic analysis of tRNA function in protein synthesis. Two genes for this tRNA species have been cloned, (Caillet et al, 1983;Schwartz et al, 1983), facilitating their localized mutagenesis. However, the isolation of mutants of Escherichia coli tRNAPhe has recently become easier.…”

Section: Introductionmentioning

confidence: 99%

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

Vacher¹,

Springer²,

Buckingham³

1985

The EMBO Journal

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The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de‐attenuation of beta‐galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe‐tRNA synthetase. Mutants showing de‐attenuation were sequenced and two nucleotide changes identified: G44‐‐‐‐A44 (found five times) and m7G46‐‐‐‐A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2‐‐‐U2 and G15‐‐‐‐A15; the mutant m7G46‐‐‐‐A46 was also re‐isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de‐attenuation is that mutant tRNAPhe molecules compete with the wild‐type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.

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Identiflcation of clones anE.colitRNA^phegene by suppression of pheylananyl-tRNA synthetase thermosensitive mutants

Cited by 19 publications

References 14 publications

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

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Identiflcation of clones anE.colitRNAphegene by suppression of pheylananyl-tRNA synthetase thermosensitive mutants

Cited by 19 publications

References 14 publications

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Expression in Escherichia coli of Bacillus subtilis tRNA genes from a promoter within the tRNA gene region

Bacillus subtilis phenylalanyl-tRNA synthetase genes: cloning and expression in Escherichia coli and B. subtilis

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

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Identiflcation of clones anE.colitRNA^phegene by suppression of pheylananyl-tRNA synthetase thermosensitive mutants