Significance of Knotted Structures for Function of Proteins and Nucleic Acids - September 17-21, 2014

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.

- 69 -