sequence while maintaining the secondary structure stability and
function.
To understand the basic concepts in folding of more complex
structures that contain more than one structural element, several
studies interrogated folding of the P5abc stem-loop structure from
the Tetrahymena thermophila ribozyme [45–47]. P5abc is a long
stem-loop structure that contains three irregularities: a nucleotide
bulge, an A-rich internal loop, and an additional hairpin, P5c,
branching off of the stem (Fig. 2a). Elimination of the internal
loop and the junctional hairpin in the P5ab construct showed a
two-state formation of P5ab, indicative of a highly cooperative
mechanism of the hairpin folding. Addition of the P5c hairpin in
P5abcΔA retained two-state folding although folding and unfolding rates decreased. This reduction in transition between states
likely results from nucleation of two hairpins instead of one; therefore, kinetic barriers for each substructure must be crossed prior to
completion of folding. Finally, in the presence of both the P5c
hairpin and A-rich bulge, the construct P5abc folds and unfolds
with intermediates. Thus, introduction of irregularities into the
regular stem-loop structure changes kinetics of folding and breaks
highly cooperative formation of the helix providing the structural
and kinetic foundation for tertiary interactions in the functional
domain.
4 RNA Tertiary Structure Formation
Despite its fundamental importance, cooperativity does not necessarily contribute to all aspects of protein folding as some regions of
proteins can fold and unfold as independent units. RNA molecules,
like proteins, must fold into three-dimensional structures to carry
out biological functions. However, RNA can form stable secondary
structures in the absence of tertiary structure, thus posing a question of whether cooperativity needs to be employed in tertiary RNA
folding. The P4-P6 domain of the Tetrahymena group I ribozyme
represents a model RNA system that has been extensively studied to
determine the extent of cooperativity in tertiary RNA folding [48–
51]. The crystal structure of the P4-P6 domain [41] revealed the
side-by-side packing of two helical structures (Fig. 2b) connected
by the J5/5a turn and stabilized by two long-distance tertiary
contacts involving the metal core/metal core receptor and the
tetraloop/tetraloop receptor [41, 52, 53]. Thus, P4-P6 domain
folding may require cooperativity between two tertiary contacts.
To gain insight into the cooperative folding mechanism, the
P4-P6 domain folding was studied using a thermodynamic box
similar to the one we described for the study of hairpin folding
[48]. Each of the tertiary contacts was disrupted by mutations, one
site at a time, and each RNA was internally labeled by two
RNA Cooperativity and Allostery
263
function.
To understand the basic concepts in folding of more complex
structures that contain more than one structural element, several
studies interrogated folding of the P5abc stem-loop structure from
the Tetrahymena thermophila ribozyme [45–47]. P5abc is a long
stem-loop structure that contains three irregularities: a nucleotide
bulge, an A-rich internal loop, and an additional hairpin, P5c,
branching off of the stem (Fig. 2a). Elimination of the internal
loop and the junctional hairpin in the P5ab construct showed a
two-state formation of P5ab, indicative of a highly cooperative
mechanism of the hairpin folding. Addition of the P5c hairpin in
P5abcΔA retained two-state folding although folding and unfolding rates decreased. This reduction in transition between states
likely results from nucleation of two hairpins instead of one; therefore, kinetic barriers for each substructure must be crossed prior to
completion of folding. Finally, in the presence of both the P5c
hairpin and A-rich bulge, the construct P5abc folds and unfolds
with intermediates. Thus, introduction of irregularities into the
regular stem-loop structure changes kinetics of folding and breaks
highly cooperative formation of the helix providing the structural
and kinetic foundation for tertiary interactions in the functional
domain.
4 RNA Tertiary Structure Formation
Despite its fundamental importance, cooperativity does not necessarily contribute to all aspects of protein folding as some regions of
proteins can fold and unfold as independent units. RNA molecules,
like proteins, must fold into three-dimensional structures to carry
out biological functions. However, RNA can form stable secondary
structures in the absence of tertiary structure, thus posing a question of whether cooperativity needs to be employed in tertiary RNA
folding. The P4-P6 domain of the Tetrahymena group I ribozyme
represents a model RNA system that has been extensively studied to
determine the extent of cooperativity in tertiary RNA folding [48–
51]. The crystal structure of the P4-P6 domain [41] revealed the
side-by-side packing of two helical structures (Fig. 2b) connected
by the J5/5a turn and stabilized by two long-distance tertiary
contacts involving the metal core/metal core receptor and the
tetraloop/tetraloop receptor [41, 52, 53]. Thus, P4-P6 domain
folding may require cooperativity between two tertiary contacts.
To gain insight into the cooperative folding mechanism, the
P4-P6 domain folding was studied using a thermodynamic box
similar to the one we described for the study of hairpin folding
[48]. Each of the tertiary contacts was disrupted by mutations, one
site at a time, and each RNA was internally labeled by two
RNA Cooperativity and Allostery
263