Abstract

Small synthetic lariat RNAs have been found to undergo site specific self-cleavage to give an acyclic branched-RNA with 2'9'-cyclic phosphate and a 5'-hydroxyl termini, which is reminiscent of the products formed in some catalytic RNAs. These lariat-RNAs are much smaller than the natural catalytic RNAs such as the hammerhead ribozyme (k = similar to 1 min(-1) at 37 degrees C), and their rate of the self-cleavage is also much slower (k = 0.25 x 10(4) min(-1) for lariat hexamer 18, and 0.16 x 10(-3) min(-1) for lariat heptamer 19 at 22 degrees C). We have shown that the trinucleotidyl loop in the tetrameric and pentameric lariat-RNAs (ref. 10) is completely stable whereas the tetranucleotidyl or pentanucleotidyl loop in the hexameric or heptameric lariat-RNA (ref. 10-13) does indeed have the required local and global conformation promoting the self-cleavage. It has been also shown that simple 2'-->5' or 3'-->5'-linked cyclic RNAs, 16 and 17, respectively, ore completely stable and their structures are considerably different from the self-cleaving lariat-RNAs such as 18 or 19. In our search to explore the optimal structural requirement for the self-cleavage reaction of RNA, we have now synthesized 14 in which the branch-point adenosine has a 2'-->5'-linked tetranucleotidyl loop and a 3'-ethylphosphate moiety mimicking the 3'-tail of the lariat-hexamer 18. We here report that the unique 3'-ethylphosphate function at the branch-point in 14 is the key structural feature that orchestrates its self-cleavage reaction (k = 0.15 x 10(4) min(-1) at 19 degrees C) compared to the stable 2'-->5'-linked cyclic RNA id (see Fig. 1). We also report the detailed conformational features of the self-cleaving tetrameric lariat-RNA 14 by 500 MHz NMR spectroscopy and Molecular dynamics simulations in the aqueous environment. A comparative study of the temperature dependence of the N reversible arrow S equilibrium for the lariat-tetramer 14 and the 2'-->5'-linked cyclic tetramer 16 shows that the A(1) residue in 14 is in 92% S-type conformation at 20 degrees C, wheras it is only in 55% S in 16 with a 3'-hydroxyl group.This displacement of the N reversible arrow S pseudorotational equilibrium toward the S geometry is due to the enhanced gauche effect of the 3'-OPO(3)Et- group at the branch-point adenosine in 14 compared to 3'-OH group in Id, This 3'-OPO(3)Et-group promoted stabilization of the S geometry al the branch-point by Delta H approximate to 4 kcal.mol(-1) in 14 is the conformational driving force promoting its unique self-cleavage reaction. The comparison of Delta H degrees and Delta S degrees of the N reversible arrow S pseudorotational equilibria in 14 and 16 (see Table 5) clearly shows the remarkable effect of the 3'-ethylphosphate group in 14 in being able to dictate the conformational changes from the sugar moiety of the branch-point adenosine to the entire molecule (conformational transmission. Thus the S conformation in A(1) U-2 and C-6 sugar moieties is clearly thermodynamically more stabilized while it is considerably destabilized in G(3) owing to the 3'-ethylphosphate group in 14 compared to 16. It is interesting to note that the magnitude of enthalpy and entropy for the North to South transition of the A(1) sugar in 14 is comparable to the enthalpy and entropy of transition between the A- and B-form of the lariat hexamer 18 (ref. 12) This self-cleaving tetrameric lariat-RNA 14 is the smallest RNA molecule hitherto known to undergo the self-cleavage reaction and hence it is the simplest model of the active cleavage site of the natural self-cleaving catalytic RNA.

Published in

Tetrahedron
1994, Volume: 50, number: 29, pages: 8711-8736
Publisher: PERGAMON-ELSEVIER SCIENCE LTD

UKÄ Subject classification

Structural Biology


Publication identifier

DOI: https://doi.org/10.1016/S0040-4020(01)85346-1

Permanent link to this page (URI)

https://res.slu.se/id/publ/83885