Antisense inhibition of Escherichia coli RNase P RNA: mechanistic aspects.
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 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. Binding 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 many 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.
Gruegelsiepe
Heike
H
Philipps-Universität Marburg, Institut für Pharmazeutische Chemie, Marbacher Weg 6, 35037 Marburg, Germany.
Willkomm
Dagmar K
DK
Goudinakis
Olga
O
Hartmann
Roland K
RK
D001412:Bacillus subtilis; D001483:Base Sequence; D002413:Cations, Divalent; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007711:Klebsiella pneumoniae; D007854:Lead; D008969:Molecular Sequence Data; D009174:Mycoplasma; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D011550:Pseudomonas aeruginosa; D012329:RNA, Bacterial / Q000138:chemical synthesis / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D016376:Oligonucleotides, Antisense; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D007854:Lead; D012343:RNA, Transfer; D043262:Ribonuclease P; C470865:ribonuclease P, E coli