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首页> 外文期刊>Journal of bacteriology >Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations.
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Structure of Escherichia coli K-12 miaA and characterization of the mutator phenotype caused by miaA insertion mutations.

机译：大肠杆菌K-12 miaA的结构以及由miaA插入突变引起的突变表型的表征。

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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.
机译：以前，我们报道了大肠杆菌K-12中2-甲硫基-N6-（δ2-异戊烯基）腺苷37（ms2i6A-37）tRNA修饰与自发诱变之间的一些异常关系（DM Connolly和ME Winkler，J. Bacteriol） 171：3233-3246，1989）。为了证实和扩展这些观察结果，我们确定了miaA的结构，该结构介导了ms2i6A-37合成的第一步，并表征了miaA突变体表型。 miaA最可能的翻译起点与mutL的最后两个密码子重叠，该密码子编码一种甲基指导的错配修复所需的蛋白质。这种结构安排证实了miaA和mutL在同一个复杂操纵子中。 miaA基因产物，δ2-异戊烯基焦磷酸转移酶，与酵母MOD5基因产物具有广泛的同源性，两种酶均含有在法尼基焦磷酸合成酶中发现的底物结合位点和一个保守的推定ATP / GTP结合位点。 miaA中的插入仅引起GC ---- TA转换，这与在mutL突变体中观察到的GC ---- AT和AT ---- GC转换形成对比。若要将不存在的ms2i6A-37 tRNA修饰直接与突变体表型相关联，我们分离出泄漏miaA（ochre）突变的独特抑制因子。 miaD抑制剂定​​位到99.75分钟，将ms2i6A-37 tRNA修饰恢复为miaA（ochre）突变体，并废除了miaA突变体表型。我们推测miaD会导致ms2i6A-37 tRNA的降低或miaA基因表达的增加，但不会导致操纵子转录水平的降低。总之，这些观察结果支持ms2i6A-37 tRNA修饰充当调节自然突变频率和其他代谢功能的生理开关的想法。

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