Binding of Helix‐Threading Peptides to E. coli 16S Ribosomal RNA and Inhibition of the S15–16S Complex

Abstract

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.

Advances in RNA structure analysis by chemical probing

“…For example, MPE (methidiumpropyl-EDTA) binds selectively to a simple bulged RNA element [49,50]. An MPE binding site, and thus a tethered Fe(II)-EDTA group, can be placed at many sites by simply mutating the sequence of an RNA helix (Fig.…”

Inhibition of Bacterial Ribosome Assembly: a Suitable Drug Target?

“…In another approach, a peptide derivative called a helixthreading peptide was found to intercalate with an RNA helix that is part of the S15 binding site. The peptide interferes with S15 binding, but whether results with this minimal system can be reproduced in vitro with the whole binding site or even the whole ribosome remains to be seen (64). More specifically, protein S15, although important for assembly in vitro, has since been found to be dispensable in vivo (17), confirming the disconnect between the processes in vitro and in vivo.…”

Native-like RNA Tertiary Structures Using a Sequence-Encoded Cleavage Agent and Refinement by Discrete Molecular Dynamics

“…tRNA sequences were embedded in the context of 5' and 3' structure cassette10 sequences. The sequence of the bulge-containing competitor RNA was 5'-GGGCGGUUUU UCGAAGCUUG AGUCUCGUCC GGGCUUCGGC CUGGACGAAG ACUGACGCUC-3' 28. RNAs were purified by denaturing polyacrylamide gel electrophoresis (PAGE), excised from the gel, and recovered by electroelution and ethanol precipitation.…”