Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations.
Previously, we reported several unusual relationships between the 2-methylthio-N6-(delta 2-isopentenyl)adenosine-37 (ms2i6A-37) tRNA modification and spontaneous mutagenesis in Escherichia coli K-12 (D. M. Connolly and M. E. Winkler, J. Bacteriol. 171:3233-3246, 1989). To confirm and extend these observations, we determined the structure of miaA, which mediates the first step of ms2i6A-37 synthesis, and characterized the miaA mutator phenotype. The most likely translation start of miaA overlaps the last two codons of mutL, which encodes a protein required for methyl-directed mismatch repair. This structural arrangement confirms that miaA and mutL are in the same complex operon. The miaA gene product, delta 2-isopentenylpyrophosphate transferase, shows extensive homology with the yeast MOD5 gene product, and both enzymes contain a substrate binding site found in farnysyl pyrophosphate synthetase and a conserved putative ATP/GTP binding site. Insertions in miaA cause exclusively GC----TA transversions, which contrasts with the GC----AT and AT----GC transitions observed in mutL mutants. To correlate the absence of the ms2i6A-37 tRNA modification directly with the mutator phenotype, we isolated a unique suppressor of a leaky miaA(ochre) mutation. The miaD suppressor mapped to 99.75 min, restored the ms2i6A-37 tRNA modification to miaA(ochre) mutants, and abolished the miaA mutator phenotype. We speculate that miaD causes a decrease in ms2i6A-37 tRNA demodification or an increase in miaA gene expression but not at the level of operon transcription. Together, these observations support the idea that the ms2i6A-37 tRNA modification acts as a physiological switch that modulates spontaneous mutation frequency and other metabolic functions.
Connolly
D M
DM
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Winkler
M E
ME
D000595:Amino Acid Sequence; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D007541:Isopentenyladenosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010641:Phenotype; D012343:RNA, Transfer / Q000235:genetics*; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013489:Suppression, Genetic
D004269:DNA, Bacterial; C008503:2-methylthio-N-6-isopentenyladenosine; D007541:Isopentenyladenosine; D012343:RNA, Transfer