Monitoring Molecular Recognition of the Ribosomal Decoding Site

Abstract

Adenin dreht sich raus: Röntgenkristallographie, Fluoreszenzmarkierung mit 2‐Aminopurin und die Verwendung von Aminoglycosiden als Liganden ermöglichten den Nachweis von Konformationsänderungen an der RNA‐Domäne, die die richtige Proteinsynthese sicherstellt (siehe Bild). Das Auslösen einer Änderung der Konformation einer RNA‐Adenineinheit durch die Bindung von Liganden kann als Grundlage für eine Methode zur Suche nach Antibiotika dienen.

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

“…Model oligonucleotides designed to mimic the drug-binding site have been used to investigate various aspects of aminoglycoside-ribosome interaction (18)(19)(20)(21)(22). However, conclusions derived from the study of model A-site oligonucleotides are compromised by several findings: (i) in contrast to drug susceptibility of complete ribosomes, binding affinities of aminoglycosides to prokaryotic decoding region constructs are not very sensitive to mutations within the RNA-binding region (23); (ii) in vivo drug susceptibilities of mutant ribosomes and in vitro binding affinities using variants of model A-site oligonucleotides may or may not correlate (24)(25)(26); (iii) the exquisite specificity of aminoglycosides for the prokaryotic as opposed to the eukaryotic cytosolic ribosome contrasts with the observation that these drugs bind to eukaryotic decoding-site constructs with approximately the same affinity as found for their prokaryotic counterpart (23,24); and (iv) while there is evidence that mitochondrial ribosomes are susceptible to aminoglycosides (13,27), oligonucleotides mimicking the mitochondrial A site do not bind aminoglycosides to any significant extent (24,28).…”

Structure-Guided Discovery of Novel Aminoglycoside Mimetics as Antibacterial Translation Inhibitors

“…1B), which are locked in one state upon binding of aminoglycoside antibiotics (4). RNA constructs of the decoding-site sequence that have either A1492 or A1493 replaced by fluorescent bases such as 2-aminopurine or 3-MI can be used to monitor ligand binding by measuring fluorescence quenching or enhancement upon titration with a potential binder (12,20). While these experiments do not necessarily reveal the exact binding place or orientation of a ligand, interaction in an aminoglycoside-like fashion can be surmised as long as RNA complex formation induces a change in the chemical environment of the fluorescent base.…”

“…Compounds were tested for binding to the decoding-site target by using an RNA fluorescence assay (20) which determines the binding affinity of a ligand based on its ability to quench or enhance emission of a fluorescent label attached at the positions of the flexible adenines A1492 or A1493 upon association with a model oligonucleotide (Fig. 3C).…”

“…The excitation wavelength was at 350 nm, while emission was monitored at 430 nm. Complete experimental details of the assay have been reported elsewhere (20).…”

Synthesis and site-specific incorporation of a simple fluorescent pyrimidine

“…Many biophysical assays have taken advantage of this feature because base flipping is a rather common event in biologically relevant nucleic acids. Selected examples include: (i) 'real-time' monitoring of the hammerhead ribozyme folding, cleavage and inhibition 34,35 , (ii) evaluating the dimerization and isomerization of the HIV-1 dimerization initiation site (DIS) 36 , (iii) examining RNA-protein interactions such as the HIV RRE-Rev binding 37,38 , and (iv) studying the binding of aminoglycoside antibiotics to the bacterial decoding site, known as the A-site 39,40 .…”