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 condons 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 miaK 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 ms2i6 A-37 tRNA modification acts as a physiological switch that modulates spontaneous mutation frequency and other metabolic functions.