Isolation of the gene (miaE) encoding the hydroxylase involved in the synthesis of 2-methylthio-cis-ribozeatin in tRNA of Salmonella typhimurium and characterization of mutants.
The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present at position 37 (3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(I,V Ser) in Escherichia coli. Salmonella typhimurium 2-methylthio-cis-ribozeatin (ms2io6A) is found in tRNA, probably in the corresponding species that have ms2i6A in E. coli. The gene (miaE) for the tRNA(ms2io6A)hydroxylase of S. typhimurium was isolated by complementation in E. coli. The miaE gene was localized close to the argI gene at min 99 of the S. typhimurium chromosomal map. Its DNA sequence and transcription pattern together with complementation studies revealed that the miaE gene is the second gene of a dicistronic operon. Southern blot analysis showed that the miaE gene is absent in E. coli, a finding consistent with the absence of the hydroxylated derivative of ms2i6A in this species. Mutants of S. typhimurium which have MudJ inserted in the miaE gene and which, consequently, are blocked in the ms2i6A hydroxylation reaction were isolated. Unexpectedly, such mutants cannot utilize the citric acid cycle intermediates malate, fumarate, and succinate as carbon sources.
Persson
B C
BC
Department of Microbiology, Umeå University, Sweden.
Björk
G R
GR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D007541:Isopentenyladenosine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016366:Open Reading Frames; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000096:biosynthesis*; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics*; D013045:Species Specificity; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003062:Codon; D017931:DNA Primers; C036211:N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine; D007541:Isopentenyladenosine; D012343:RNA, Transfer; C586270:2-methylthio-N6-isopentenyl-adenosine(37)-tRNA monooxygenase, Salmonella typhimurium; D006899:Mixed Function Oxygenases