A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.
We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.
Department of Genetics, University of Leicester, UK.
M X
MX
Chen
N
N
Bouquin
V
V
Norris
S
S
Casarégola
S J
SJ
Séror
I B
IB
Holland
D001483:Base Sequence; D015139:Blotting, Southern; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003062:Codon; D015342:DNA Probes; D004269:DNA, Bacterial / Q000096:biosynthesis; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012356:RNA, Transfer, Leu / Q000235:genetics*; D015183:Restriction Mapping; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism; D003189:p-Methoxy-N-methylphenethylamine / Q000494:pharmacology*
D002147:Calmodulin; D003062:Codon; D015342:DNA Probes; D004269:DNA, Bacterial; D012356:RNA, Transfer, Leu; D003189:p-Methoxy-N-methylphenethylamine; D001616:beta-Galactosidase