Madsen Christian Toft, Mengel-Jørgensen Jonas, Kirpekar Finn, Douthwaite Stephen
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
Nucleic Acids Res. 2003 Aug 15;31(16):4738-46. doi: 10.1093/nar/gkg657.
There are three sites of m(5)U modification in Escherichia coli stable RNAs: one at the invariant tRNA position U54 and two in 23S rRNA at the phylogenetically conserved positions U747 and U1939. Each of these sites is modified by its own methyltransferase, and the tRNA methyltransferase, TrmA, is well-characterised. Two open reading frames, YbjF and YgcA, are approximately 30% identical to TrmA, and here we determine the functions of these candidate methyltransferases using MALDI mass spectrometry. A purified recombinant version of YgcA retains its activity and specificity, and methylates U1939 in an RNA transcript in vitro. We were unable to generate a recombinant version of YbjF that retained in vitro activity, so the function of this enzyme was defined in vivo by engineering a ybjF knockout strain. Comparison of the methylation patterns in 23S rRNAs from YbjF(+) and YbjF(-) strains showed that the latter differed only in the lack of the m(5)U747 modification. With this report, the functions of all the E.coli m(5)U RNA methyltransferases are identified, and a more appropriate designation for YbjF would be RumB (RNA uridine methyltransferases B), in line with the recent nomenclature change for YgcA (now RumA).
在大肠杆菌稳定RNA中存在三个5-甲基尿苷（m(5)U）修饰位点：一个位于不变的tRNA位置U54，另外两个位于23S rRNA中系统发育保守位置U747和U1939。这些位点中的每一个都由其自身的甲基转移酶进行修饰，并且tRNA甲基转移酶TrmA已得到充分表征。两个开放阅读框YbjF和YgcA与TrmA的序列相似度约为30%，在此我们使用基质辅助激光解吸电离质谱法（MALDI质谱法）来确定这些候选甲基转移酶的功能。纯化的重组YgcA保留了其活性和特异性，并在体外使RNA转录本中的U1939甲基化。我们无法产生具有体外活性的重组YbjF版本，因此通过构建一个ybjF基因敲除菌株在体内定义了该酶的功能。对来自YbjF(+)和YbjF(-)菌株的23S rRNA甲基化模式的比较表明，后者仅在缺乏m(5)U747修饰方面有所不同。随着本报告的发表，所有大肠杆菌m(5)U RNA甲基转移酶的功能均已确定，并且根据YgcA（现称为RumA）最近的命名变化，YbjF更合适的名称应为RumB（RNA尿苷甲基转移酶B）。
