Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase
Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase
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DOI：
10.1074/jbc.m704572200
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发表时间：
2007-09-21
影响因子：
4.8
通讯作者：
Appling, Dean R.
中科院分区：
文献类型：
--
作者：
Lee, Changkeun;Kramer, Gisela;Appling, Dean R.
TRM5基因编码酿酒酵母胞质tRNA中37(m(1)G37)位鸟苷甲基化的tRNA(鸟氨酸-N1-)甲基转移酶(Trm5p)。在这里，我们证明了Trm5p也与线粒体tRNAs的m(1)G37甲基化有关。TRMS开放阅读框编码499个氨基酸，在前48个密码子中包含4个潜在的启动子密码子。全长Trm5p是一种与麦芽糖结合蛋白的融合蛋白，它与从Delta trm5突变株分离的tRNA以及合成的线粒体启动子tRNA(tRNA(F)(Met))具有很强的甲基转移酶活性。扩增结果表明，线粒体tRNA(F)(Met)和tRNA(Ph)的甲基化位点均为鸟苷37。高压液相色谱分析表明，甲基化产物为m(1)G。亚细胞分离和免疫印迹分析表明，该酶定位于细胞质和线粒体。稍大的线粒体形式被保护，不被蛋白酶消化，这表明基质定位。对N-末端截断突变体的分析表明，在缺少氨基酸1-33(Delta 1-33)的情况下，可以获得在细胞质中活性的Trm5p，而在线粒体中活性Trm5p的产生需要这前33个氨基酸。表达Delta 1-33结构的酵母表现出显著较低的耗氧率，这表明在缺乏线粒体tRNAs m(1)G37甲基化的细胞中，线粒体蛋白质合成的效率或准确性降低。这些数据表明，这种tRNA修饰在线粒体蛋白质合成的阅读框架维持中起着重要作用。
The TRM5 gene encodes a tRNA (guanine-N1-)-methyttransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRMS open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Delta trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA(f)(Met)). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA(f)(Met) and tRNA(Ph). High pressure liquid chromatography analysis showed the methylated product to be m(1)G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRMS gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1-33 (Delta 1-33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Delta 1-33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m(1)G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis.