Molecular cloning and sequencing ofpheU, a gene forEscherichia colitRNA^Phe

Abstract

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.

Precise physical mapping of theEscherichia coli pheU transcription unit

“…Under our conditions, this fragment directs the synthesis of a l l0-nucleotide-long RNA which probably corresponds to a transcript starting at the putative promoter and terminating at the putative rho-independent terminator. The restriction map of the pEB I0 plasmid is homolo; gous to that one of pMU393 [6] and of the plD2 [7] except the inversion of a 3.6 kb Sall fragment to be in accordance with the Escherichia coli chromosomal map [8]. The nucleotide sequence is identical to that one described [6,9] for the tRNA ~' )' gone mapped to 94 min.…”

“…The 3,6 kb Sa/l fragment in pID2 (shadcd box) has been inverscd from that shown in [7]. The restriction .~itcs arc: B, BamHl: H, H/ndlII; R. EcoRl; E, EcoRV; Bg, Bg/h P, Pstl: S. Sa/1; A, Avah C, Clal.…”

“…Here we provide details of the physical and genetic mapping ofpheU, its transcription unit and confirm its nucleotide sequence. The genes phe V and pheU were characterized by four independent groups ( [6,7,9] and this work). The gene pheU, called pheR in [6], was cloned using a screen based on the control ofpheA expression.…”

“…Both pheU and pheV were found in a systematic screen for tRNA carrying phages fi'om the ordered Kohara library [9]. However, one library gave two clones, called phel'V [11] and pheU [7], with abberant sequences which probably derive from pheU. Sall restriction fragment of pCS8 has been used in in vitro transcription experiments (Fig.…”

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

“…(i) They served as hosts in the initial subcloning and determination of the relative locations of genes in a DNA region covering min 37 of the E. coli linkage map (1), which includes the PheRS loci pheS and pheT for the a and ,B subunits, respectively (30,35). (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).…”