title: Helix 69 of Escherichia coli 23S ribosomal RNA as a peptide nucleic acid target.
abstract: A fragment of 23S ribosomal RNA (nucleotides 1906-1924 in E. coli), termed Helix 69, forms a hairpin that is essential for ribosome function. Helix 69 forms a conformationally flexible inter-subunit connection with helix 44 of 16S ribosomal RNA, and the nucleotide A1913 of Helix 69 influences decoding accuracy. Nucleotides U1911 and U1917 are post-transcriptionally modified with pseudouridines (Ψ) and U1915 with 3-methyl-Ψ. We investigated Helix 69 as a target for a complementary synthetic oligonucleotide - peptide nucleic acid (PNA). We determined thermodynamic properties of Helix 69 and its complexes with PNA and tested the performance of PNA targeted at Helix 69 in inhibiting translation in cell-free extracts and growth of E. coli cells. First, we examined the interactions of a PNA oligomer complementary to the G1907-A1919 fragment of Helix 69 with the sequences corresponding to human and bacterial species (with or without pseudouridine modifications). PNA invades the Helix 69 hairpin creating stable complexes and PNA binding to the pseudouridylated bacterial sequence is stronger than to Helix 69 without any modifications. Second, we confirmed the binding of PNA to 23S rRNA and 70S ribosomes. Third, we verified the efficiency of translation inhibition of these PNA oligomers in the cell-free translation/transcription E. coli system, which were in a similar range as tetracycline. Next, we confirmed that PNA conjugated to the (KFF)3K transporter peptide inhibited E. coli growth in micromolar concentrations. Overall, targeting Helix 69 with PNA or other sequence-specific oligomers could be a promising way to inhibit bacterial translation.
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Faculty of Chemistry, University of Warsaw, Pasteura 1, 02-093, Warsaw, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Kulik
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland; Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096, Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Markowska-Zagrajek
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Wojciechowska
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
authors.forename: Renata
authors.initials: R
authors.lastname: Grzela
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Wituła
authors.affiliation: Centre of New Technologies, University of Warsaw, Banacha 2c, 02-097, Warsaw, Poland. Electronic address: joanna@cent.uw.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Trylska
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects*; D009690:Nucleic Acid Conformation; D020135:Peptide Nucleic Acids / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D011560:Pseudouridine; D012329:RNA, Bacterial / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D020135:Peptide Nucleic Acids; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D011560:Pseudouridine
keywords: 23S ribosomal RNA; Bacterial growth inhibition; Cell-free translation; Helix 69; Peptide nucleic acid; RNA hairpin