Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena.
We have developed a quantitative substitution interference technique to examine the role of Pro-Rp oxygens in the phosphodiester backbone of RNA, using phosphorothioates as a structural probe. This approach is generally applicable to any reaction involving RNA in which the precursor and reaction products can be separated. We have applied the technique to identity structural requirements in the group I intron from Tetrahymena thermophila for catalysis of hydrolysis at the 3' splice site; 44 phosphate oxygens are important in 3' splice site hydrolysis. These include four or five oxygens previously observed to be important in exon ligation. Although phosphate oxygens having a functional significance can be found throughout the intron, the strongest phosphorothioate effects are closely associated with positions in the highly conserved intron core, which are likely to be involved in tertiary interactions, substrate recognition and catalysis.
Christian
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Department of Molecular Cellular and Developmental Biology, University of Colorado, Boulder 80309-0347.
Yarus
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D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010100:Oxygen / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism*; D012326:RNA Splicing; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016808:Tetrahymena thermophila / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology; D014764:Viral Proteins
D010710:Phosphates; D016053:RNA, Protozoan; D013873:Thionucleotides; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D010100:Oxygen