Antisense inhibition of Escherichia coli RNase P RNA:: Mechanistic aspects
Antisense inhibition of Escherichia coli RNase P RNA:: Mechanistic aspects
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DOI：
10.1002/cbic.200300675
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发表时间：
2003-10-06
期刊：
影响因子：
3.2
通讯作者：
Hartmann, RK
中科院分区：
文献类型：
--
作者：
Gruegelsiepe, H;Willkomm, DK;Hartmann, RK
核糖核蛋白酶RNase P在生命的所有领域中催化tRNA初级转录物的内切核酸5 '-成熟。RNase P对细菌细胞生长的不可或缺性以及细菌和真核生物RNase P酶之间在结构和功能上的巨大差异符合细菌酶适合作为潜在的新型药物靶标的基本要求。我们已经鉴定了RNA寡核苷酸，其在我们的体外前体tRNA（ptRNA）加工测定中在亚纳摩尔浓度下开始显示对结构类型A的细菌RNase P RNA（例如，大肠杆菌或肺炎克雷伯氏菌酶）的抑制作用。这些寡核苷酸针对已知与ptRNA底物的3 '-CCA部分相互作用的RNase P RNA的所谓P15环区。铅探测实验表明，一个互补的RNA或DNA 14聚体完全侵入P15环区，从而破坏局部结构的催化核心的RNase P RNA。RNA 14-mer的结合基本上是不可逆的，因为解离速率非常低。该寡核苷酸的结合速率为10（4）M-1 s（-1）的量级，因此与许多其它人工反义寡核苷酸的结合速率相当。显着的抑制效果是由于直接干扰与RNase P RNA活性位点结合的底物和诱导RNase P RNA催化核心的错误折叠的双重作用。基于我们的研究结果，结构类型A的细菌RNase P RNA的P15环区域可以被认为是核酶的“阿喀琉斯之踵”，因此代表了对抗多重耐药细菌病原体的有希望的靶标。
The ribonucleoprotein enzyme RNase P catalyzes endonucleolytic 5'-maturation of tRNA primary transcripts in all domains of life. The indispensability of RNase P for bacterial cell growth and the large differences in structure and function between bacterial and eukaryotic RNase P enzymes comply with the basic requirements for a bacterial enzyme to be suitable as a potential novel drug target. We have identified RNA oligonucleotides that start to show an inhibitory effect on bacterial RNase P RNAs of the structural type A (for example, the Escherichia coli or Klebsiella pneumoniae enzymes) at a subnanomolar concentrations in our in vitro precursor tRNA (ptRNA) processing assay. These oligonucleotides are directed against the so-called P15 loop region of RNase P RNA known to interact with the 3'-CCA portion of ptRNA substrates. Lead probing experiments demonstrate that a complementary RNA or DNA 14-mer fully invades the P15 loop region and thereby disrupts local structure in the catalytic core of RNase P RNA. Bindin of the RNA 14-mer is essentially irreversible because of a very low dissociation rate. The association rate of this oligonucleotide is on the order of 10(4) M-1 s(-1) and is thus comparable to those of may other artificial antisense oligonucleotides. The remarkable inhibition efficacy is attributable to the dual effect of direct interference with substrate binding to the RNase P RNA active site and induction of misfolding of the catalytic core of RNase P RNA. Based on our findings, the P15 loop region of bacterial Rnase P RNAs of the structural type A can be considered the "Achilles' heel" of the ribozyme and therefore represents a promising target for combatting multiresistant bacterial pathogens.