Determination of the Hill coefficient relies on measuring binding affinity between RNA and ligands and does not require deep
knowledge of the system’s thermodynamics or special knowledge
from the experimenter beyond the ability to employ a binding
technique. The Hill coefficient can be determined by a variety of
approaches including both the techniques used for studying
protein-ligand interactions and the methods that exploit unique
chemical and structural properties of RNA. The former includes
various spectroscopic methods and ITC, as discussed earlier.
Among the latter, it is worth mentioning techniques developed to
probe ligand-induced changes in the conformation and stability of
RNA molecules, such as in-line probing [29], nuclease cleavage
[20], and chemical probing [30, 31]. Although all techniques aim
to detect ligand-induced allosteric changes in RNA, in-line probing
is probably the most robust and easiest method that does not
require incubation with specific probes (nucleases or chemicals)
and special treatments to stop the cleavage or modification reaction. In-line probing specifically exploits inherent instability of
RNA in water solutions, which is more pronounced in flexible
regions and greatly accelerated by divalent Mg
2+ cations and elevated pH [32]. The method involves incubation of end-labeled
RNA molecules in the absence of the ligand and at various ligand
concentrations, and detection of changes in the RNA cleavage
pattern after separating RNA fragments electrophoretically on a
denaturing polyacrylamide gel. The extent of changes upon ligand
titration can be used as a measure of binding affinity and the Hill
coefficient. The method is naturally restricted to probe the RNA
regions that change stability by binding to a ligand or becoming
involved in intermolecular interactions as a result of ligand binding.
3 Folding of RNA into Its Secondary Structure
Although RNA is capable of forming intricate tertiary structures
paralleled to those formed by proteins, the folding of both macromolecules involves different forces and results in dissimilar structural features. In contrast to proteins that have twenty amino acids,
RNA is composed of only four similar chemical blocks with 50%
more atoms than a protein having the same number of residues.
Along with a larger size, RNA contains more dihedral bonds where
rotations that introduce a greater potential for alternative conformations can occur. Unlike proteins, which are mostly composed of
α-helices and β-sheets merged into a compact structure by hydrophobic interactions of side chains, RNA predominantly adopts a
single secondary structure element, the double-stranded helix. This
structural element contains many negative charges made up of
phosphate groups on its periphery, thereby restricting the assembly
of a hydrophobic core. Formation of helices in RNA is mostly
RNA Cooperativity and Allostery
261
knowledge of the system’s thermodynamics or special knowledge
from the experimenter beyond the ability to employ a binding
technique. The Hill coefficient can be determined by a variety of
approaches including both the techniques used for studying
protein-ligand interactions and the methods that exploit unique
chemical and structural properties of RNA. The former includes
various spectroscopic methods and ITC, as discussed earlier.
Among the latter, it is worth mentioning techniques developed to
probe ligand-induced changes in the conformation and stability of
RNA molecules, such as in-line probing [29], nuclease cleavage
[20], and chemical probing [30, 31]. Although all techniques aim
to detect ligand-induced allosteric changes in RNA, in-line probing
is probably the most robust and easiest method that does not
require incubation with specific probes (nucleases or chemicals)
and special treatments to stop the cleavage or modification reaction. In-line probing specifically exploits inherent instability of
RNA in water solutions, which is more pronounced in flexible
regions and greatly accelerated by divalent Mg
2+ cations and elevated pH [32]. The method involves incubation of end-labeled
RNA molecules in the absence of the ligand and at various ligand
concentrations, and detection of changes in the RNA cleavage
pattern after separating RNA fragments electrophoretically on a
denaturing polyacrylamide gel. The extent of changes upon ligand
titration can be used as a measure of binding affinity and the Hill
coefficient. The method is naturally restricted to probe the RNA
regions that change stability by binding to a ligand or becoming
involved in intermolecular interactions as a result of ligand binding.
3 Folding of RNA into Its Secondary Structure
Although RNA is capable of forming intricate tertiary structures
paralleled to those formed by proteins, the folding of both macromolecules involves different forces and results in dissimilar structural features. In contrast to proteins that have twenty amino acids,
RNA is composed of only four similar chemical blocks with 50%
more atoms than a protein having the same number of residues.
Along with a larger size, RNA contains more dihedral bonds where
rotations that introduce a greater potential for alternative conformations can occur. Unlike proteins, which are mostly composed of
α-helices and β-sheets merged into a compact structure by hydrophobic interactions of side chains, RNA predominantly adopts a
single secondary structure element, the double-stranded helix. This
structural element contains many negative charges made up of
phosphate groups on its periphery, thereby restricting the assembly
of a hydrophobic core. Formation of helices in RNA is mostly
RNA Cooperativity and Allostery
261