Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli
Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli
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DOI：
10.1128/jb.181.23.7256-7265.1999
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发表时间：
1999-12-01
影响因子：
3.2
通讯作者：
Winkler, ME
中科院分区：
文献类型：
--
作者：
Esberg, B;Leung, HCE;Winkler, ME
鼠伤寒沙门氏菌miaB2508：：Tn10dCm突变株的tRNA缺乏修饰核苷N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine(ms(2)io(6)A37)的甲硫基。通过测序，我们发现该菌株的Tn10dCm已插入到f474(YleA)的开放阅读框中，该阅读框位于鼠伤寒沙门氏菌和大肠杆菌中的NAG位点附近。通过miaB2508：：Tn10dCm突变与最小的亚克隆f474片段互补，我们证明f474是MIAB基因，在细菌染色体上以逆时针方向转录。转录研究发现，在大肠杆菌和鼠伤寒沙门氏菌中，MIAB的上游有两个启动子。在MIAB基因下游发现了一个不依赖于Rho的终止子，大多数(96%)MIAB转录本在该终止子上终止于大肠杆菌，这表明MIAB基因是单顺反子操纵子的一部分。在MIAB中存在一个高度保守的基序，含有三个半胱氨酸残基。这个基序类似于其他蛋白质中的铁结合位点。仅发现与ADOMet结合位点的微弱相似性，支持MIAB蛋白参与硫代化步骤而不参与ms(2)I(O)(6)A37形成的甲基化反应的想法。
The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleoside N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508:: Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream of miaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of the miaB gene, at which the majority (96%) of the miaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation.