Abstract

The small ''lariat'' hexameric 1 and heptameric 2 RNAs undergo self-cleavage, whereas the two cyclic A(2'-->5)G and A(3'-->5')G linked tetramers 3 and 4 do not self-cleave. The site of phosphodiester cleavage is specific and occurs at the 3'-phosphate of the guanosine residue to give a guanosine 2',3'-cyclic phosphate and a 5'-hydroxyl termini. At 22 degrees C, the heptamer 2 (k = 0.16 x 10(-3) min(-1)) cleaves ca. six times faster than the hexamer 1 (k = 0.25 x 10(-4) min(-1)). The rare of cleavage is temperature and pH dependent. The addition of Mg2+ ions slightly increases the rate of cleavage, but NMR studies show that it does not produce any changes in the conformation of the ribose rings and of the glycosidic bonds. H-1-NMR shows that the lariat-hexamer 1 exists as two conformers (A and B) in slow exchange on the NMR time scale. The enthalpy term (Delta H = 7.1 kcal mol(-1)) favours the A-form while the entropy term (Delta S = 21 cal mol(-1) K-1) favours the B-form. The energy of activation for the transition between the A- and B-forms is 23 kcal.mol(-1). The loop nucleotides in the B-form of hexamer 1 have ribose, glycosidic bonds and phosphate backbone conformation that are very similar to those of heptamer 2. At low temperature, the conformation of the A(2'-->5)G linked tetramer 3 and A(3'-->5')G linked tetramer 4 is very similar to the conformation of the A-form of hexamer 1. Torsional constraints derived from H-1-H-1, H-1-P-31 and C-13-P-31 coupling constants were used for molecular dynamics simulations in water with sodium counterions for a total of 226 ps. The MD simulations were first carried out with torsional constraints derived from J-couplings (0-96 ps) and then completely without constraints (106-226 ps). No major conformational changes occurred upon the release of the constraints indicating that the ensemble of conformers generated during the MD simulation are not artificially held in these conformations by the NMR constraints and these conformers may be good representatives of the actual NMR observed solution structures. A comparison of the self-cleavage rate between hexamer, heptamer and hammerhead-RNA (k(cat) approximate to 0.5 min(-1) at 37 degrees C) also suggests that the cleavage-site geometry of the hammerhead-RNA should be much closer to the transition state/intermediate geometry than heptamer 2. The pH-dependent study of the self-cleavage reaction of hexamer hexamer 1 has shown that the self-cleavage rate peaks at pH 6 and slows down considerably both above and below this pH. Nonspecific cleavage starts becoming important at a very low pH (less than or equal to 3) and at a high pH (greater than or equal to 10). The structures generated during both NMR constrained and unconstrained MD runs show that the cleavage-site between G(3) and U-7 in heptamer 2 has the following average local conformation: S-sugar for G(3), epsilon(-) (-86 degrees+/-13 degrees), zeta(+) (91 degrees+/-16 degrees),alpha(+) (85 degrees+/-24 degrees), beta(t) (163 degrees+/-13 degrees) and gamma(+) (65 degrees+/-11 degrees). Molecular modelling studies on the MD generated geometry show that a simple rotation of the local phosphate backbone at the cleavage-site from epsilon(-) (d(O2'-3'P) = 3.8 Angstrom) --> epsilon = 120 degrees (d(O2'-3'P) = 2.8 Angstrom) and a rotation of zeta(+) --> zeta(t) would position the leaving 5'-terminus of U-7 for a potential in-line displacement by 2'-OH of G(3) (Note that in the latter geometrical transition, alpha(+), beta(t) and gamma(+) and the South-sugar of U-7 remain unchanged). Such a geometry at the cleavage site would produce an optimal local structure for a neighbouring nucleophilic attack by 2'-OH to give the trigonal-bipyramidal phosphorane transition state/intermediate.

Published in

Tetrahedron
1994, Volume: 50, number: 6, pages: 18101777 Publisher: PERGAMON-ELSEVIER SCIENCE LTD

UKÄ Subject classification

Structural Biology


Publication identifier

DOI: https://doi.org/10.1016/S0040-4020(01)80852-8

Permanent link to this page (URI)

https://res.slu.se/id/publ/83888