Exploring purine N7 interactions via atomic mutagenesis: The group I ribozyme as a case study

Abstract

Atomic mutagenesis has emerged as a powerful tool to unravel specific interactions in complex RNA molecules. An early extensive study of analogs of the exogenous guanosine nucleophile in group I intron self-splicing by Bass and Cech demonstrated structure-function relationships analogous to those seen for protein ligands and provided strong evidence for a wellformed substrate binding site made of RNA. Subsequent functional and structural studies have confirmed these interacting sites and extended our understanding of them, with one notable exception. Whereas 7-methyl guanosine did not affect reactivity in the original study, a subsequent study revealed a deleterious effect of the seemingly more conservative 7-deaza substitution. Here we investigate this paradox, studying these and other analogs with the more thoroughly characterized ribozyme derived from the Tetrahymena group I intron. We found that the 7-deaza substitution lowers binding by~20-fold, relative to the cognate exogenous guanosine nucleophile, whereas binding and reaction with 7-methyl and 8-aza-7-deaza substitutions have no effect. These and additional results suggest that there is no functionally important contact between the N7 atom of the exogenous guanosine and the ribozyme. Rather, they are consistent with indirect effects introduced by the N7 substitution on stacking interactions and/or solvation that are important for binding. The set of analogs used herein should be valuable in deciphering nucleic acid interactions and how they change through reaction cycles for other RNAs and RNA/protein complexes.

Multiple RNA–RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome

“…The double-deaza mutant (U6-7cA59/7cG60) led to the accumulation of splicing intermediates (Figure 3). Model studies have shown that deaza substitutions in a helix negatively affect helix stability by reducing stacking, hydration and/or cation interaction (58,59). The double-deaza mutant would therefore be predicted to lead to weakened base stacking, suggesting that the A59/G60 stack in the U2/U6 RNA core (Figure 5) might have a stabilising effect on the structural transitions between steps 1 and 2 of splicing.…”

Impact of 3-deazapurine nucleobases on RNA properties

“…This can be determining for RNA base pairing ( 8 , 9 ), for RNA recognition of other nucleic acids (e.g. DNA, 2′-OCH 3 RNA) ( 6 ), proteins ( 3 ), small molecules ( 8 ), and ions ( 9 ), and this can also be crucial with respect to RNA-catalyzed reactions ( 4 , 10–13 ). Concerning the latter, atomic mutagenesis lead to our current in-depth understanding of the chemical mechanism of phosphodiester cleavage of the recently discovered twister and pistol ribozymes ( 14–16 ).…”

“…Suitable deazanucleosides for informative RNA atomic mutagenesis experiments are 7-deazaadenosine (c 7 A) ( 4 , 11–13 , 15 , 16 ), 3-deazaadenosine (c 3 A) ( 14 , 20 ), 1-deazaadenosine (c 1 A) ( 11 , 12 , 14 , 20 ), 7-deazaguanosine (c 7 G) ( 13 , 15 ) and 3-deazacytidine (c 3 C) ( 13 , 21 ). Moreover, 3-deazaguanosine (c 3 G) and 1-deazaguanosine (c 1 G) would be highly useful for RNA atomic mutagenesis, however, such studies are very rare ( 22 ) because synthetic access to appropriate phosphoramidite building blocks and to the corresponding RNAs is challenging ( 22 , 23 ).…”

Structural and Energetic Impact of Non‐natural 7‐Deaza‐8‐azaguanine, 7‐Deaza‐8‐azaisoguanine, and Their 7‐Substituted Derivatives on Hydrogen‐Bond Pairing with Cytosine and Isocytosine

“…[19] Finally, in the context of RNA, DAG has been used as an analog to map out the interactions of the exogenous guanosine molecule in the Tetrahymena group I ribozyme, and shown to compromise neither its binding nor its reactivity. [20] Figure 1. Structure of (a) guanine (G); (b) 7-substituted-7-deaza-8-azaguanine (7-X-DAG); (c) isoguanine (iG); (d) 7-substituted-7-deaza-8-aza-isoguanine (7-X-DAiG) with conventional numbering of purines.…”