Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations

Abstract

Previously, we reported several unusual relationships between the 2-methylthio-N6-(A2-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, A2-isopentenylpyrophosphate transferase, shows extensive homology with the yeast MODS 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.The hypermodified, hydrophobic 2-methylthio-N6-(A2-isopentenyl)adenosine (ms2i6A) modification (Fig.

Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis

“…ophimuritlm, Strep. pneumoniae and Axotobacter vinelandii, confirmed the functional similarities. The MutS and HexA proteins are highly similar, and so are MutL and HexB (Connoly & Winkler, 1992;Haber e t al., 1988;Le e t a/., 1993;Mankovich e t al., 1989;Prudhomme e t al., 1989Prudhomme e t al., , 1991Priebe e t al., 1988 ;Schlensong & Boeck, 1991). Negative complementation conferred by the expression in E. coli of the Strep, pneumonzae HexA function confirmed their common evolutionary origin (Prudhomme e t al., 1991).…”

Enzymatic Modification of tRNAs

“…On the other hand tRNAs from mutant strains lacking a functional miaB gene have been shown to contain only i 6 A-37, the product of the first step of the pathway suggesting that the MiaB protein is involved in C-S bond formation (17). Deficiency in methylthiolation leads to a decreased efficiency of the corresponding tRNAs and an increased spontaneous mutation frequency (18,19).…”

An editing-defective aminoacyl-tRNA synthetase is mutagenic in aging bacteria via the SOS response

“…The paucity of AT 3 GC mutations suggests that a loss of MutS activity is not a significant contributor to the MAC phenotype in this case. Finally, two known mechanisms (mutYM or miaA) may be responsible for the increased frequency of GC 3 TA mutations (12,(16)(17)(18). In addition, these two mechanisms may be functioning to differing extents in the editing-defective and wild-type strains.…”