Defining the Chemical Groups Essential for Tetrahymena Group I Intron Function by Nucleotide Analog Interference Mapping

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1997 Apr 1

PMID 9096319

Citations 29

Authors

S A Strobel

K Shetty

Affiliations

Soon will be listed here.

Abstract

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.

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References

Been M, Cech T . Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence. Cell. 1987; 50(6):951-61. DOI: 10.1016/0092-8674(87)90522-8. View

Tanner M, Cech T . Joining the two domains of a group I ribozyme to form the catalytic core. Science. 1997; 275(5301):847-9. DOI: 10.1126/science.275.5301.847. View

Barfod E, Cech T . The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing. Mol Cell Biol. 1989; 9(9):3657-66. PMC: 362426. DOI: 10.1128/mcb.9.9.3657-3666.1989. View

Doudna J, Cormack B, Szostak J . RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron. Proc Natl Acad Sci U S A. 1989; 86(19):7402-6. PMC: 298070. DOI: 10.1073/pnas.86.19.7402. View

Waring R . Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution. Nucleic Acids Res. 1989; 17(24):10281-93. PMC: 335300. DOI: 10.1093/nar/17.24.10281. View

Cech T . Self-splicing of group I introns. Annu Rev Biochem. 1990; 59:543-68. DOI: 10.1146/annurev.bi.59.070190.002551. View

Green R, Ellington A, Szostak J . In vitro genetic analysis of the Tetrahymena self-splicing intron. Nature. 1990; 347(6291):406-8. DOI: 10.1038/347406a0. View

Ruffner D, Uhlenbeck O . Thiophosphate interference experiments locate phosphates important for the hammerhead RNA self-cleavage reaction. Nucleic Acids Res. 1990; 18(20):6025-9. PMC: 332400. DOI: 10.1093/nar/18.20.6025. View

Michel F, Westhof E . Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol. 1990; 216(3):585-610. DOI: 10.1016/0022-2836(90)90386-Z. View

10.

Celander D, Cech T . Visualizing the higher order folding of a catalytic RNA molecule. Science. 1991; 251(4992):401-7. DOI: 10.1126/science.1989074. View

11.

Green R, Szostak J, Benner S, Rich A, Usman N . Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron. Nucleic Acids Res. 1991; 19(15):4161-6. PMC: 328556. DOI: 10.1093/nar/19.15.4161. View

12.

Dahm S, Uhlenbeck O . Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry. 1991; 30(39):9464-9. DOI: 10.1021/bi00103a011. View

13.

Moore M, Sharp P . Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992; 256(5059):992-7. DOI: 10.1126/science.1589782. View

14.

Pyle A, Murphy F, Cech T . RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme. Nature. 1992; 358(6382):123-8. DOI: 10.1038/358123a0. View

15.

Beaudry A, Joyce G . Directed evolution of an RNA enzyme. Science. 1992; 257(5070):635-41. DOI: 10.1126/science.1496376. View

16.

Christian E, Yarus M . Analysis of the role of phosphate oxygens in the group I intron from Tetrahymena. J Mol Biol. 1992; 228(3):743-58. DOI: 10.1016/0022-2836(92)90861-d. View

17.

Piccirilli J, Vyle J, Caruthers M, Cech T . Metal ion catalysis in the Tetrahymena ribozyme reaction. Nature. 1993; 361(6407):85-8. DOI: 10.1038/361085a0. View

18.

Gaur R, Krupp G . Modification interference approach to detect ribose moieties important for the optimal activity of a ribozyme. Nucleic Acids Res. 1993; 21(1):21-6. PMC: 309060. DOI: 10.1093/nar/21.1.21. View

19.

Wang J, Downs W, Cech T . Movement of the guide sequence during RNA catalysis by a group I ribozyme. Science. 1993; 260(5107):504-8. DOI: 10.1126/science.7682726. View

20.

Christian E, Yarus M . Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry. 1993; 32(17):4475-80. DOI: 10.1021/bi00068a001. View