Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly
Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly
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DOI：
10.1128/jb.186.17.5819-5825.2004
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发表时间：
2004-09-01
影响因子：
3.2
通讯作者：
Dahlberg, AE
中科院分区：
文献类型：
--
作者：
Cameron, DM;Gregory, ST;Dahlberg, AE
细菌中的核糖体蛋白L11被PRMA基因的产物L11甲基转移酶在多个氨基酸位置翻译后三甲基化。L11甲基化在核糖体功能或组装中的作用尚未确定，尽管大肠杆菌PRMA的缺失没有明显的表型。我们构建了极端嗜热菌Thermus thermophilus的突变株，其中PrMA基因已经被编码耐热卡那霉素腺基转移酶的HTK基因打乱。该突变体没有表现出生长缺陷，这表明嗜热T菌和它的大肠杆菌同源物一样，是可有可无的。经基质辅助激光解吸电离飞行时间质谱仪(MALDI-TOF MS)鉴定，该突变株制备的核糖体含有未甲基化的L11，是克隆和纯化嗜热梭菌体外甲基化的有效底物。NLkLDI-TOF MS还揭示了嗜热链霉菌L11总共含有12个甲基，而在大肠杆菌L11中发现了9个甲基。最后，我们发现，与大肠杆菌甲基转移酶一样，从核糖体分离出来的核糖体蛋白L11是PRMA比完整的70S核糖体更有效的体外甲基化底物，这表明体内甲基化发生在游离L11上，而不是整合到核糖体中。
The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase. This mutant shows no growth defects, indicating that T. thermophilus PrmA, like its E. coli homolog, is dispensable. Ribosomes prepared from this mutant contain unmethylated L11, as determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and are effective substrates for in vitro methylation by cloned and purified T. thermophilus PrmA. NLkLDI-TOF MS also revealed that T. thermophilus L11 contains a total of 12 methyl groups, in contrast to the 9 methyl groups found in E. coli L11. Finally, we found that, as with the E. coli methyltransferase, the ribosomal protein L11 dissociated from ribosomes is a more efficient substrate for in vitro methylation by PrmA than intact 70S ribosomes, suggesting that methylation in vivo occurs on free L11 prior to its incorporation into ribosomes.