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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-(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.

SUBMITTER: Connolly DM

PROVIDER: S-EPMC207322 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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

Connolly D M DM Winkler M E ME

Journal of bacteriology 19910301 5

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 ...[more]

PMID: 1999389

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Project description:UnlabelledMotility is a beneficial attribute that enables cells to access and explore new environments and to escape detrimental ones. The organelle of motility in Escherichia coli is the flagellum, and its production is initiated by the activating transcription factors FlhD and FlhC. The expression of these factors by the flhDC operon is highly regulated and influenced by environmental conditions. The flhDC promoter is recognized by σ(70) and is dependent on the transcriptional activator cyclic AMP (cAMP)-cAMP receptor protein complex (cAMP-CRP). A number of K-12 strains exhibit limited motility due to low expression levels of flhDC. We report here a large number of mutations that stimulate flhDC expression in such strains. They include single nucleotide changes in the -10 element of the promoter, in the promoter spacer, and in the cAMP-CRP binding region. In addition, we show that insertion sequence (IS) elements or a kanamycin gene located hundreds of base pairs upstream of the promoter can effectively enhance transcription, suggesting that the topology of a large upstream region plays a significant role in the regulation of flhDC expression. None of the mutations eliminated the requirement for cAMP-CRP for activation. However, several mutations allowed expression in the absence of the nucleoid organizing protein, H-NS, which is normally required for flhDC expression.ImportanceThe flhDC operon of Escherichia coli encodes transcription factors that initiate flagellar synthesis, an energetically costly process that is highly regulated. Few deregulating mutations have been reported thus far. This paper describes new single nucleotide mutations that stimulate flhDC expression, including a number that map to the promoter spacer region. In addition, this work shows that insertion sequence elements or a kanamycin gene located far upstream from the promoter or repressor binding sites also stimulate transcription, indicating a role of regional topology in the regulation of flhDC expression.

Dataset Information

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

Publications

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

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