a large distance, ligands bind to the RNA in cooperative manner
under physiological concentrations of Mg
2+ cations (Fig. 3b).
Binding of both ligands is required for stabilization of tertiary
interactions which ensure large allosteric changes resulting in the
formation of a transcription terminator instead of an antiterminator
in the downstream regions. Ablation of binding by mutagenesis in
one site decreases binding affinity to the second site, thus suggesting that binding to one site facilitates ligand interactions with the
other. Mutation analyses further indicates that one site is more
important for genetic control than the other. High Mg
2+ concentrations apparently provide extra stabilization to the structure and
diminish cooperativity between ligand-binding sites.
The c-di-AMP riboswitch [61–63] is another example of double ligand binding to a single RNA domain. Interestingly, the
riboswitch adopts a twofold pseudosymmetrical square that binds
two molecules of c-di-AMP along opposite sides of the square in
almost identical fashion. Although cooperativity has not been
directly determined, mutagenesis studies have shown that tertiary
structure formation requires binding of both ligands [61]. Elimination of one ligand-binding site reduces ligand binding to the second ligand, suggesting that formation of one binding pocket is
required for long-distance allosteric change that causes the
subsequent folding of the other pocket. These data further suggest
cooperative binding of two ligands, a hypothesis awaiting confirmation by further biochemical and biophysical studies.
5.2 Allosteric
Changes Define
Cooperative Assembly
of RNA-Protein
Complexes
RNA participates in the formation of many ribonucleoprotein
(RNP) complexes involved in mRNA processing, localization,
transport, translation, and other cellular processes. One of the
best studied RNPs is the ribosome, which consists of two subunits,
30S and 50S, formed by several dozen proteins and a few RNA
molecules. The 30S subunit can be assembled from purified proteins and RNA; however, the reconstitution of the functional subunit requires a specific order for protein binding, suggestive of
multiple allosteric modulations and a cooperative manner of assembly [64]. The central domain of the ribosome initially forms separately from the rest of the subunit and, therefore, represents an
excellent model system to study cooperativity and associated allosteric modulations. Structural and biochemical studies revealed that
the domain assembly involves highly cooperative binding of ribosomal proteins [22, 65–68]. The process is initiated by binding of
the ribosomal protein S15 to a ~200 nt region of 16S rRNA
(Fig. 3c). The binding re-arranges and stabilizes conformations of
two three-helix junctions. The top junction constitutes the binding
site for the dimer of proteins S6 and S18; therefore, S15 binding
facilitates further binding of the S6:S18 complex [22, 68]. Thermodynamic studies showed that binding of S15 and S6:S18 heterodimer is highly cooperative, with coupling free energy of
RNA Cooperativity and Allostery
267
under physiological concentrations of Mg
2+ cations (Fig. 3b).
Binding of both ligands is required for stabilization of tertiary
interactions which ensure large allosteric changes resulting in the
formation of a transcription terminator instead of an antiterminator
in the downstream regions. Ablation of binding by mutagenesis in
one site decreases binding affinity to the second site, thus suggesting that binding to one site facilitates ligand interactions with the
other. Mutation analyses further indicates that one site is more
important for genetic control than the other. High Mg
2+ concentrations apparently provide extra stabilization to the structure and
diminish cooperativity between ligand-binding sites.
The c-di-AMP riboswitch [61–63] is another example of double ligand binding to a single RNA domain. Interestingly, the
riboswitch adopts a twofold pseudosymmetrical square that binds
two molecules of c-di-AMP along opposite sides of the square in
almost identical fashion. Although cooperativity has not been
directly determined, mutagenesis studies have shown that tertiary
structure formation requires binding of both ligands [61]. Elimination of one ligand-binding site reduces ligand binding to the second ligand, suggesting that formation of one binding pocket is
required for long-distance allosteric change that causes the
subsequent folding of the other pocket. These data further suggest
cooperative binding of two ligands, a hypothesis awaiting confirmation by further biochemical and biophysical studies.
5.2 Allosteric
Changes Define
Cooperative Assembly
of RNA-Protein
Complexes
RNA participates in the formation of many ribonucleoprotein
(RNP) complexes involved in mRNA processing, localization,
transport, translation, and other cellular processes. One of the
best studied RNPs is the ribosome, which consists of two subunits,
30S and 50S, formed by several dozen proteins and a few RNA
molecules. The 30S subunit can be assembled from purified proteins and RNA; however, the reconstitution of the functional subunit requires a specific order for protein binding, suggestive of
multiple allosteric modulations and a cooperative manner of assembly [64]. The central domain of the ribosome initially forms separately from the rest of the subunit and, therefore, represents an
excellent model system to study cooperativity and associated allosteric modulations. Structural and biochemical studies revealed that
the domain assembly involves highly cooperative binding of ribosomal proteins [22, 65–68]. The process is initiated by binding of
the ribosomal protein S15 to a ~200 nt region of 16S rRNA
(Fig. 3c). The binding re-arranges and stabilizes conformations of
two three-helix junctions. The top junction constitutes the binding
site for the dimer of proteins S6 and S18; therefore, S15 binding
facilitates further binding of the S6:S18 complex [22, 68]. Thermodynamic studies showed that binding of S15 and S6:S18 heterodimer is highly cooperative, with coupling free energy of
RNA Cooperativity and Allostery
267