fluorophores, Cy3 and Cy5, which produce FRET, when coming in
close proximity. Measuring FRET at the single-molecule level
allowed detection of the folded state and determination of equilibrium constants for structural transitions in the wild-type and two
mutant RNAs. Quantification of tertiary cooperativity from these
measurements revealed that in each case the tertiary contact formation is 240-fold more favorable subsequent to formation of the
other tertiary contact. Therefore, RNA folding may involve cooperative formation of tertiary contacts, at least in the systems with
close positioning of such contacts.
5 RNA-Ligand Interactions
5.1 Cooperativity
and Allostery
in Binding of Ligands
to Riboswitches
Cooperativity in RNA interactions occurs beyond folding of secondary and tertiary structures. Many RNAs interact with various
cations and some RNAs bind small molecules or macromolecules in
a cooperative manner. Examples of such RNAs include riboswitches, noncoding RNA regions which are capable of undergoing
large allosteric transitions to modulate expression of the adjacent
genes in response to specific binding to cellular metabolites and
ions (reviewed in [54]). Evolutionarily conserved metabolitesensing domains of riboswitches adopt intricate three-dimensional
structures that specifically recognize cognate metabolites and reject
similar compounds.
The cognate ligands of the M-box riboswitch, found in Bacillus
subtilis mgtE gene, are multiple Mg
2+ cations [55]. Mg
2+ cations
are among the most abundant divalent cations in cells and are
Fig. 2 Cooperative interactions in folding of the P4-P6 domain of the Tetrahymena group I ribozyme. (a)
Folding of the P5 stem-loop structure [45]. Molecular constructs used for assessing the folding of the RNA are
on the left and the entire domain is on the right. (b) Molecular construct used to determine cooperativity of
tertiary contact formation [48, 49]. Tertiary contacts are depicted in colors and connected by arrows. Internal
fluorescent labels and FRET are shown with stars and a green arrow, respectively
264
Alla Peselis and Alexander Serganov
close proximity. Measuring FRET at the single-molecule level
allowed detection of the folded state and determination of equilibrium constants for structural transitions in the wild-type and two
mutant RNAs. Quantification of tertiary cooperativity from these
measurements revealed that in each case the tertiary contact formation is 240-fold more favorable subsequent to formation of the
other tertiary contact. Therefore, RNA folding may involve cooperative formation of tertiary contacts, at least in the systems with
close positioning of such contacts.
5 RNA-Ligand Interactions
5.1 Cooperativity
and Allostery
in Binding of Ligands
to Riboswitches
Cooperativity in RNA interactions occurs beyond folding of secondary and tertiary structures. Many RNAs interact with various
cations and some RNAs bind small molecules or macromolecules in
a cooperative manner. Examples of such RNAs include riboswitches, noncoding RNA regions which are capable of undergoing
large allosteric transitions to modulate expression of the adjacent
genes in response to specific binding to cellular metabolites and
ions (reviewed in [54]). Evolutionarily conserved metabolitesensing domains of riboswitches adopt intricate three-dimensional
structures that specifically recognize cognate metabolites and reject
similar compounds.
The cognate ligands of the M-box riboswitch, found in Bacillus
subtilis mgtE gene, are multiple Mg
2+ cations [55]. Mg
2+ cations
are among the most abundant divalent cations in cells and are
Fig. 2 Cooperative interactions in folding of the P4-P6 domain of the Tetrahymena group I ribozyme. (a)
Folding of the P5 stem-loop structure [45]. Molecular constructs used for assessing the folding of the RNA are
on the left and the entire domain is on the right. (b) Molecular construct used to determine cooperativity of
tertiary contact formation [48, 49]. Tertiary contacts are depicted in colors and connected by arrows. Internal
fluorescent labels and FRET are shown with stars and a green arrow, respectively
264
Alla Peselis and Alexander Serganov