Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli
Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli
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DOI：
10.1074/jbc.m111825200
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发表时间：
2002-03-15
影响因子：
4.8
通讯作者：
Stroud, RM
中科院分区：
文献类型：
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作者：
Agarwalla, S;Kealey, JT;Stroud, RM
大肠杆菌开放阅读框ygcA被鉴定为推定的23 S核糖体RNA 5-甲基尿苷甲基转移酶（Gustafsson，C.，里德河，格林，P. J.，和Santi，D.（1996）Nucleic Acids Res.24，3756-3762）。我们已经克隆，表达和纯化的50 kDa的蛋白质编码的ygcA。纯化的酶催化23 S rRNA的甲基化，但不作用于16 S rRNA或tRNA。基于高效液相色谱的核苷分析鉴定反应产物为5-甲基尿苷。通过核酸酶保护试验和使用23 S rRNA位点特异性突变体的甲基化试验确定酶特异性甲基化U1939。一个40个核苷酸的23 S rRNA片段（核苷酸1930-1969）也作为酶的有效底物。40-mer RNA寡核苷酸和α-Met的表观Km值分别为3和26 gm，表观k（cat）为0.06 s（-1）。该酶含有两个当量的铁/单体，并具有与铁硫蛋白中发现的基序相似的序列基序。我们建议将该基因命名为鲁马，并相应地将蛋白质产物命名为鲁马，即RNA尿苷甲基转移酶。
An Escherichia coli open reading frame, ygcA, was identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762). We have cloned, expressed, and purified the 50-kDa protein encoded by ygcA. The purified enzyme catalyzed the AdoMet-dependent methylation of 23 S rRNA but did not act upon 16 S rRNA or tRNA. A high performance liquid chromatography-based nucleoside analysis identified the reaction product as 5-methyluridine. The enzyme specifically methylated U1939 as determined by a nuclease protection assay and by methylation assays using site-specific mutants of 23 S rRNA. A 40-nucleotide 23 S rRNA fragment (nucleotide 1930-1969) also served as an efficient substrate for the enzyme. The apparent K-m values for the 40-mer RNA oligonucleotide and AdoMet were 3 and 26 gm, respectively, and the apparent k(cat) was 0.06 s(-1). The enzyme contains two equivalents of iron/monomer and has a sequence motif similar to a motif found in iron-sulfur proteins. We propose to name this gene rumA and accordingly name the protein product as RumA for RNA uridine methyltransferase.