Significance of Knotted Structures for Function of Proteins and Nucleic Acids
Poster Session I
21 – POS
Board 21
Binding of Peptide Nucleic Acid Oligomers to Helix 69 of 23S Ribosomal RNA
Marta Kulik
, Agnieszka Markowska-Zagrajek, Tomasz Witula, Joanna Trylska.
University of Warsaw, Warsaw, Poland.
Targeting a fragment of bacterial rRNA called helix 69 (H69) with complementary peptide
nucleic acid (PNA) oligomers seems to be a promising way to inhibit bacterial translation in a
sequence-specific manner. H69 is a well conserved hairpin of 23S rRNA which influences
initiation and accuracy of translation, peptidyl transferase reaction and ribosome recycling. From
the structural point of view, H69 is a part of an intersubunit bridge B2a, which contacts the D
stems of A- and P-site tRNAs. Deletion of H69 results in dominant lethal phenotype [1]. Several
studies allowed to identify the residues A1912, U1917 and A1919 of H69 as essential for
ribosomal functioning [2,3].
Based on the above mutational studies and accessibility of H69 in the ribosome structure we
have designed a 13-nucleotide long PNA oligomer covering the sequence stretch G1907 –
A1919 and investigated its interactions with isolated H69 hairpin. We performed melting
temperature measurements, isothermal titration calorimetry, circular dichroism spectroscopy and
non-denaturing gel electrophoresis to investigate the structural properties of the PNA-H69
complexes. Two PNA variants were tested: with and without a cell penetrating peptide (KFF)
3
K.
The PNA interactions with H69 of
E. coli
sequence were compared with the corresponding
sequence in human ribosomes.
Results show that both PNAs invade the RNA loop and create stable complexes with H69.
Binding of PNA to
E. coli
H69 was stronger than to human sequence. We have also confirmed
the efficiency of translation inhibition of the PNA oligomers in cell-free
E. coli
extracts.
References:
[1] Ali et al.,
Molecular Cell
, 2006, 23, 865–874.
[2] Hirabayashi et al.,
J. Biol. Chem
., 2006, 281, 25.
[3] Kipper et al.,
J. Mol. Biol
., 2009, 385, 405–422.
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