Lariat formation in splicing of pre‐messenger RNA. Conformation and base stacking at the lariat branch point studied using 500‐MHz 1H NMR and CD spectroscopy

Abstract

AbstractA conformational‐analysis study based on 500‐MHz 1H NMR and circular dichroism (CD) has been performed on the branched trinucleotide U‐5′‐3′‐A‐2′‐5′‐G (1) which models the branch point in the lariat structure of Group‐II excised introns. The NMR and CD results are in agreement with previous work in which it was shown that adenine 2′‐5′ guanine base stacking essentially determines the molecular conformation of compound 1. The ribose ring of the A residue shows a preference for the S conformation, while the riboses of U and G are highly flexible, i.e. no clear preference for N or S is observed. CD spectroscopy of 1 showed that ethylene glycol leads to disruption of adenine 2′‐5′ guanine stacking. Furthermore, we studied the unnatural branched trinucleotide G‐5′‐3′‐A‐2′‐5′‐U (2) in which U and G are reversed in comparison with compound 1. The molecular conformation of 2 is far more rigid in comparison with 1, as a result of very strong adenine 2′‐5′ uracil stacking. Ethylene glycol was apparently unable to facilitate de‐stacking of adenine and uracil in 2. The present results show that guanosine as the 2′linked nucleotide corresponds with a clear molecular flexibility. This may be of relevance in understanding why guanosine, as the 2′‐5′ nucleotide, is a prerequisite for the occurrence of the second step in group‐II splicing.