Ligand‐ and pH‐Induced Conformational Changes of RNA Domain Helix 69 Revealed by 2‐Aminopurine Fluorescence

Abstract

Schleifenkonformation: Die Schleife der RNA‐Domäne Helix‐69 (H69) wurde mit dem fluoreszierenden Adenin‐Analogon 2‐Aminopurin (2AP) modifiziert, was die je nach den Bedingungen unterschiedlichen Konformationszustände sichtbar machte. Die Verwendung dieser Modell‐RNA zeigt den besonderen Einfluss des Aminoglycosids Neomycin auf die Schleifenkonformation von H69, der sich von dem der strukturell ähnlichen Verbindungen Paromomycin und Gentamicin unterscheidet (siehe Bild).

Modulation of conformational changes in helix 69 mutants by pseudouridine modifications

“…These results suggest that Ψ modifications dominate the stabilizing effects of certain conformational states of H69 and the loop mutants. The hypothesis is supported by fluorescence studies employing modified H69 with 2-aminopurine (2AP) at position 1913, in which the fluorescence intensity from 2AP in ΨΨΨ decreased at pH 5.5 relative to 7.0 due to enhanced base stacking of 2AP1913 with neighboring bases [19]. A similar change was not observed with 2AP-containing UUU, suggesting that the modification plays a role in the conformational switch.…”

“…Since Ψ1917 and Ψ1911 do not have direct interactions in either the solution structure of ΨΨΨ [8] or X-ray crystal structures of complete ribosomes [36], the difference in spectral features between ΨΨC-Ψ1917C and ΨΨΨ at both pH conditions potentially results from an altered structure of the ΨΨC-Ψ1917C loop region caused by the mutation. Long-range effects of the U/Ψ1917C mutation on the stem-region structure, which are independent of the Ψ modifications, suggest stem-loop crosstalk [19, 41]. Mutations at key loop residues, such as 1917, involved in mediating this crosstalk, may also be responsible for monitoring interactions with the P-site tRNA and other translational factors, e.g.…”

“…Conformational modulation by Ψ was discovered by examining pH sensitivity of the H69 structure [18–19]. More specifically, an A1913 stacked structure with protection from solvent (referred to as the “closed” conformation) was observed only in pseudouridylated H69 at low pH (5.5).…”

“…In contrast, an alternate structure occurred at high pH (7.0), with increased exposure of A1913 (“open” conformation) [20–21]. These structures were proposed to be correlated with different conformational states of H69 during translation and establishment of intersubunit bridge B2a in complete ribosomes [18–19]. Pseudouridine modifications in H69 are not essential for bacterial growth under normal conditions [22]; however, loss of these modifications is disadvantageous for the growth of yeast under certain environmental challenges [23].…”

Incorporating modified bases and base-backbone linkers into PNAs for targeting RNA secondary structures

“…A fluorescent 2-aminopurine residue was incorporated into dsRNAs (Figure2.11) for the fluorescence measurements. It is known that the fluorescence signal of 2-aminopurine is sensitive to its local structural environment24, 35,[80][81] . We observed quenching of the 2-aminopurine fluorescence intensity upon the binding of PNA P11 (Figure2.12a-d, Figure2.13), which is consistent with our previous studies24, 35 .…”