Defining the chemical groups essential for Tetrahymena group I intron function 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 alphaS) in a nucleotide analog interference mapping (NAIM) assay that makes it possible to simultaneously, yet individually, determine the contribution of almost every N2 exocyclic amine of G within a large RNA. Using IMP alphaS, we identified the exocyclic amines that are essential for 5' or 3' exon ligation by the Tetrahymena group I intron. We report that the amino groups of three phylogenetically conserved guanosines (G111, G112, and G303) are important for 3' exon ligation. The amine of G22, as well as the amines of the other four 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 and G303A point mutations was detected at the site of mutation by NAIM using 5'-O-(1-thio)diaminopurine riboside monophosphate (DMP alphaS), an adenosine analog that has an N2 exocyclic amine. The G22A point mutant could also be rescued by incorporation of DMP alphaS at A24. By analogy to genetics, there are interference phenotypes comparable to loss of function, reversion, and suppression. This method can be readily extended to other nucleotide analogs for the analysis of chemical groups essential to a variety of RNA and DNA activities.
Strobel
S A
SA
Department of Biochemistry and Molecular Biophysics, Yale University, New Haven, CT 06520, USA. strobel@csb.yale.edu
Shetty
K
K
D000818:Animals; D005091:Exons; D006151:Guanosine / Q000235:genetics; D007288:Inosine / Q000235:genetics; D007438:Introns; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013768:Tetrahymena / Q000235:genetics*; D013873:Thionucleotides
D013873:Thionucleotides; D006151:Guanosine; D007288:Inosine