Probing ribosomal protein-RNA interactions with an external force.
Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites--i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations--i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by "one-side" clamping.
Laboratoire Nanobiophysique, Ecole Supérieure de Physique et Chimie Industrielles de la Ville de Paris, Unité Mixte de Recherche 7083 du Centre National de la Recherche Scientifique, 10 rue Vauquelin, 75005 Paris, France.
Pierre
P
Mangeol
Thierry
T
Bizebard
Claude
C
Chiaruttini
Marc
M
Dreyfus
Mathias
M
Springer
Ulrich
U
Bockelmann
D015335:Molecular Probes; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*
D015335:Molecular Probes; D016601:RNA-Binding Proteins; D012313:RNA