Kink-Switch Summary (in progress)
A Kink-switch is composed of 2 stems (WC and NC). The relative orientations of the two stems can vary, resulting in either bent or straight structures. We call the bent state the "Tripped State". The linear state is the "Cocked State". We consider this to be an RNA mouse-trap, We have identified, in the database (rr0033), three Tripped Kink-Switches and seven Cocked Kink-Switches. The cocked and tripped states are distinct in that no intermedatiates between them have been identified.

Polymorphism. Each of the two states, cocked and tripped, is an probably an ensemble of states. There is most clearly polymorism in the tripped state. That floppiness can be observed by comparing the seven tripped structures and can be seen in solution (Lilley) and in MD simulations (Spooner). Therefore each of the two global states consists of a series of substates.

Relationship of Kink-Switch to Kink-Turn. The cocked state of a Kink-Switch was previously defined as the Kink-turn (Moore). One of our contributions here is the realization via torsion-matching, that the Kink-Turn is only half of the Kink-Switch story.

The general cock-trip mechanism is shown in the Cock/Trip Global Figure. In this figure, the two strands are labeled + (plus, red line) and - (minus, black line). The figure also illustrates our generalized numbering scheme, which is used to specify residue positions. The conformational state of each residue is also specified, via the ascii code. In the cocked state, both the plus and minus strands are identified by the conformational word "aaaaraa" (the minus strands of the two parallel Kink-Switches are exceptions, see below). In the tripped state the plus strand is identified by the conformational word "aedcraa". The r-conformation of G(7+) is conserved, in both the cocked and tripped states. The conformation of the minus strand is more variable in the tripped state, although G(3-) is nearly always in the r-conformation.

(1) The NC stem of a Kink-Switch, in both the cocked and tripped states, contains a 'Hypertwist'. An axial view of a Hypertwist is shown here. A Hypertwist, one of the defining elements of a Kink-Switch, is intrinsic to the cock-trip mechanism. All Hypertwists contain the following elements:

(i) Two adjacent G-A base pairs. Residue A(4-) is paired with G(7+). Residue G(3-) is paired with A(8+), where +/- indicates the + and - strands.

(ii) An unusually high helical twist (i.e., a hypertwist) at the A(4-)-G(7+) to G(3-)-A(8+) step.

(iii) Two Hydroxyl Anchors. A Hydroxyl Anchor is a hydrogen bonding system linking the 2'hydroxyl of one guanosine and Watson-Crick face of the cross-strand guanosine upon which it is stacked. Specifically, in Hydroxyl Anchor #1 the 2'-hydroxyl group of G(7+) forms hydrogen bonds to the Watson-Crick face of G(3-) and to the phosphate group of A(8+). In Hydroxyl Anchor #2 the 2'-hydroxyl group of G(3-) forms hydrogen bonds to the Watson-Crick face of G(7+) and to the phosphate group of A(4-). The 2-amino groups of G(3-) and G(7+) are hydrogen bond donors in Hydroxyl Anchors #1 and #2, respectively.

A Hydroxyl Anchor requires the r-conformational state, which directs the C2' to O2' bond roughly parallel to the helical axis. In the rare cases where G(3-) is in a-conformation (HT-2T and HT 2501C) rather than r-conformation, Hydroxyl Anchor #2 is abolished. Hydroxyl Anchor #1 and the r-conformation of G(7+) are conserved in all hypertwists.

Requirements for Hydroxyl Anchor #1 are the r-conformational state of G(7+) to place 2'-hydroxyl group at the correct position, hypertwisting (high helical twist) from G(7+) to A(8+) to place G(3-) in correct position, and a G at position 3-, to place the 2-amino group of residue 3- at the correct position. Hypertwisting facilitates cross-strand stacking of G(7+) on G(3-) and stacking of A(4-) on A(8+).

(iv) The WC/NC interface. This interface is highly variable. Most intimate interface (8 hydrogen bonds) is observed Kink-Switch 6tr. The least intimate interface is observed in Kink-Switch 8tf (2 hydrogen bonds). (v) Extra-Helical Residues. (2) Switch Mechanism (not done) We believe that the WC-NC interface is a critical features governing the Cock-Tripped equilibrium.

(3) Strand topology. There are two fundamentally different types of hypertwist; parallel and antiparallel. The parallel class is much less probable (only two observations (1t-par and 3t-par) and will not be discussed further. However it should be noted that most of the base-base interactions and the conformation of the + strand are conserved in both parallel and antiparallel hypertwists. Therefore these should be considered a common conformational family, although with two different strand topologies.

>Solution Experiments

Use the following series of RNA oligos
8+ (control: + strand of HT 8t) (red 8)
5' C A U G C A G G A A G U C G 3'

8+(a) (+ strand of HT 8t, G* = 2' flouroG or 2'deoxyG) (red 8a )
5' C A U G C A G G* A A G U C G 3'

8+(b) (insert G1602.5 (between 1602 and 1603) (red 8b)
5' C A U G C -G- A G G A A G U C G 3'

8- (control: - strand of HT 8t) (black 8)

5' C G A U G G A A G C A U G 3'

8-(a) (remove A1591) (black 8a)
5' C G A U G G A G C A U G 3'

8-(b) (remove G1588) (black 8b)
5' C G A U G A A G C A U G 3'

black 8c (insert C1590.5 between 1590 and 1591) 5' C G A U G G A A G C A U G 3'



Predictions/Observations

wild type red strand
r8/b8 (wild type)
prediction - structure is bent/polymorphic, as per previous publications)

r8/b8A (removal of NC cap)
prediction - stabilize bend: obvervation - stabilize bend

r8/b8B (bow the NC stem)
prediction - stabilize bend: observation - stabilize bend

r8/8C (potential pair with G1604)
prediction - stabilize linear: observation - stabilize bend

DeoxyG at 1605, should lock the switch (open or closed)
r8A/b8 (wild type)
prediction - structure is bend or linear/not monomorphic: observation - structure is linear/not polymorphic

r8A/b8A (removal of NC cap)
prediction - bent, not polymorphic: obvervation - bent, not polymorphic

r8A/b8B (bow the NC stem)
prediction - bent, not polymorphic: obvervation - bent, not polymorphic

r8A/8C
prediction - bent, not polymorphic: obvervation - bent, not polymorphic