Structure modulation of helix 69 from Escherichia coli 23S ribosomal RNA by pseudouridylations

Abstract

Helix 69 (H69) is a 19-nt stem-loop region from the large subunit ribosomal RNA. Three pseudouridine (Ψ) modifications clustered in H69 are conserved across phylogeny and known to affect ribosome function. To explore the effects of Ψ on the conformations of Escherichia coli H69 in solution, nuclear magnetic resonance spectroscopy was used to reveal the structural differences between H69 with (ΨΨΨ) and without (UUU) Ψ modifications. Comparison of the two structures shows that H69 ΨΨΨ has the following unique features: (i) the loop region is closed by a Watson–Crick base pair between Ψ1911 and A1919, which is potentially reinforced by interactions involving Ψ1911N1H and (ii) Ψ modifications at loop residues 1915 and 1917 promote base stacking from Ψ1915 to A1918. In contrast, the H69 UUU loop region, which lacks Ψ modifications, is less organized. Structure modulation by Ψ leads to alteration in conformational behavior of the 5' half of the H69 loop region, observed as broadening of C1914 non-exchangeable base proton resonances in the H69 ΨΨΨ nuclear magnetic resonance spectra, and plays an important biological role in establishing the ribosomal intersubunit bridge B2a and mediating translational fidelity.

Structure of the E. coli ribosome–EF-Tu complex at <3 Å resolution by Cs-corrected cryo-EM

“…The binding site of aminoglycosides in helix 44 of 16S rRNA (h44)-including N4 of m 5 C1407-is indicated for neomycin B (magenta, superposition from PDB ID: 2ET4) 68 . The three pseudouridines stabilizing H69 69 by enhancing base stacking 70 are depicted in blue. The methyl group (yellow) on m 3 Y1917 in H69 prevents potential base-pairing with A1913, a residue important for uniform tRNA selection 71 .…”

The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes

“…Although the uridine and pseudouridine isomers are structurally very similar, they impact the conformation of H69 such that differences in the loop region occur. 22 Therefore, different binding selectivities were expected to reflect interactions of the peptides with the H69 loop region. Previous studies employed a custom-synthesized wild-type H69 RNA construct (Ψm 3 ΨΨ) for selection and binding studies, but the more readily available ΨΨΨ construct without the methylated residue was shown previously to have a similar thermal stability and secondary structure 23 and in this work to have similar affinity to the parent peptide 1 (Table 1).…”

Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

“…X-ray crystallography and solution-state nuclear magnetic resonance (NMR) spectroscopy revealed that bacterial ( Escherichia coli ) H69 assumes a hairpin structure, with a stem composed of five base pairs and a single-stranded loop of nine nucleotides [7–8], which is also observed in eukaryotic H69 [9]. Similarity in the 3D structures was deduced by high conservation and covariance of H69 sequences throughout phylogeny (Fig.…”

“…In E. coli , a single pseudouridine synthase, RluD, catalyzes this conversion at all three nucleotides in H69 [16]. The Ψ modifications have been shown to modulate structure and conformational behavior of H69 by stabilizing the loop-closing base pair and promoting base stacking in the 3′ half of the loop [8]. In E. coli H69, no net effects on hairpin stability due to Ψ modifications were observed at neutral pH.…”