DEFINING THE CHEMICAL GROUPS ESSENTIAL FOR TETRAHYMENA GROUP-I INTRONFUNCTION BY NUCLEOTIDE ANALOG INTERFERENCE MAPPING

Improved atomic resolution biochemical methods are needed to identify the chemical groups within an RNA that are essential to its activity, As a step toward this goal, we report the use of 5'-O-(1-thio)inosine monophosphate (IMP alpha S) in a nucleotide analog interference mappin g (NAIM) assay that makes it possible to simultaneously, yet individua lly, determine the contribution of almost every N2 exocyclic amine of G within a large RNA. Using IMP alpha S, we identified the exocyclic a mines that are essential for 5' or 3' exon ligation by the Tetrachymen a group I intron, We report that the amino groups of three phylogeneti cally conserved guanosines (G111, G112, and G303) are important for 3' exon ligation, The amine of G22, as well as the amines of the other f our guanosines within the P1 helix, are essential for ligation of the 5' exon. Previous work has shown that point mutation of either G22 or G303 to an adenosine (A) substantially reduces activity, Like inosine, adenosine lacks an N2 amino group, Interference rescue of the G22A an d G303A point mutations was detected at the site of mutation by NAIM u sing 5'-O-(1-thio)diaminopurine riboside monophosphate (DMP alpha S), an adenosine analog that has an N2 exocyclic amine, The G22A point mut ant could also be rescued by incorporation of DMP alpha S at A24. By a nalogy to genetics, there are interference phenotypes comparable to lo ss of function, reversion, and suppression, This method can be readily extended to other nucleotide analogs for the analysis of chemical gro ups essential to a variety of RNA and DNA activities.