IDENTIFYING RNA MINOR-GROOVE TERTIARY CONTACTS BY NUCLEOTIDE ANALOG INTERFERENCE MAPPING WITH N-2-METHYLGUANOSINE

Nucleotide analogue interference mapping (NAIM) is a general biochemic al method that rapidly identifies the chemical groups important for RN A function. In principle, NAIM can be extended to any nucleotide that can be incorporated into an in vitro transcript by an RNA polymerase. Here we report the synthesis of 5'-O-(1-thio)-N-2-methylguanosine trip hosphate (m(2)G alpha S) and its incorporation into two reverse splici ng forms of the Tetrahymena group I intron using a mutant form of T7 R NA polymerase. This analogue replaces one proton of the N2 exocyclic a mine with a methyl group, but is as stable as guanosine (G) for second ary structure formation. We have identified three sites of m(2)G alpha S interference within the Tetrahymena intron: G22, G212, and G303. Al l three of these guanosine residues are known to utilize their exocycl ic amino groups to participate in tertiary hydrogen bonds within the r ibozyme structure. Unlike the interference pattern with the phosphorot hioate of inosine (I alpha S, an analogue that deletes the N2 amine of G), m(2)G alpha S substitution did not cause interference at position s attributable to secondary structural stability effects. Given that t he RNA minor groove is likely to be widely used for helix packing, m(2 )G alpha S provides an especially valuable reagent to identify RNA min or groove tertiary contacts in less well-characterized RNAs.