Abstract
The catalytic center in group I RNAs contains a selective binding site that accommodates both guanosine and L-arginine. In order to understand the specificity of the RNA for small molecules, we analyzed 6 RNAs that vary in this region. Specificity for nucleotides resides substantially in G264 rather than its paired nucleotide C311, and is expressed substantially in Km, with comparatively little variation in kcat. kcat is not notably perturbed even for RNAs with mispairs in the active-site helix. For 5 of 6 sequences, effects of RNA substitutions on arginine binding and GTP reactivity are proportional, confirming that arginine contacts a subset of the groups occupied by G. As a result of particular mutations, reaction with GTP is decreased, and reaction with the natural nucleotides UTP and ATP is enhanced. Molecular modeling of these effects suggests that exceptionally flexible placement of reactants may be an essential quality of RNA-catalyzed splicing. The specificity of the intron can be rationalized by a type of binding model not previously considered, in which the G/arginine site includes adjacent nucleotides (an 'axial' site), rather than a single nucleotide, G264.
Full text
PDF







Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bass B. L., Cech T. R. Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor. Biochemistry. 1986 Aug 12;25(16):4473–4477. doi: 10.1021/bi00364a001. [DOI] [PubMed] [Google Scholar]
- Been M. D., Cech T. R. Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence. Cell. 1987 Sep 11;50(6):951–961. doi: 10.1016/0092-8674(87)90522-8. [DOI] [PubMed] [Google Scholar]
- Benner S. A., Ellington A. D., Tauer A. Modern metabolism as a palimpsest of the RNA world. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7054–7058. doi: 10.1073/pnas.86.18.7054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cech T. R. Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review. Gene. 1988 Dec 20;73(2):259–271. doi: 10.1016/0378-1119(88)90492-1. [DOI] [PubMed] [Google Scholar]
- Doudna J. A., Szostak J. W. RNA-catalysed synthesis of complementary-strand RNA. Nature. 1989 Jun 15;339(6225):519–522. doi: 10.1038/339519a0. [DOI] [PubMed] [Google Scholar]
- Hemmes P. R., Oppenheimer L., Jordan F. Ultrasonic relaxation evaluation of the thermodynamics of syn-anti glycosidic isomerization in adenosine. J Am Chem Soc. 1974 Sep 18;96(19):6023–6026. doi: 10.1021/ja00826a010. [DOI] [PubMed] [Google Scholar]
- Hermes J. D., Blacklow S. C., Knowles J. R. Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme. Proc Natl Acad Sci U S A. 1990 Jan;87(2):696–700. doi: 10.1073/pnas.87.2.696. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Herschlag D., Cech T. R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry. 1990 Nov 6;29(44):10159–10171. doi: 10.1021/bi00496a003. [DOI] [PubMed] [Google Scholar]
- Hicke B. J., Christian E. L., Yarus M. Stereoselective arginine binding is a phylogenetically conserved property of group I self-splicing RNAs. EMBO J. 1989 Dec 1;8(12):3843–3851. doi: 10.1002/j.1460-2075.1989.tb08562.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holbrook S. R., Sussman J. L., Warrant R. W., Kim S. H. Crystal structure of yeast phenylalanine transfer RNA. II. Structural features and functional implications. J Mol Biol. 1978 Aug 25;123(4):631–660. doi: 10.1016/0022-2836(78)90210-3. [DOI] [PubMed] [Google Scholar]
- Inoue T., Sullivan F. X., Cech T. R. Intermolecular exon ligation of the rRNA precursor of Tetrahymena: oligonucleotides can function as 5' exons. Cell. 1985 Dec;43(2 Pt 1):431–437. doi: 10.1016/0092-8674(85)90173-4. [DOI] [PubMed] [Google Scholar]
- Inoue T., Sullivan F. X., Cech T. R. New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing. J Mol Biol. 1986 May 5;189(1):143–165. doi: 10.1016/0022-2836(86)90387-6. [DOI] [PubMed] [Google Scholar]
- Kay P. S., Inoue T. Catalysis of splicing-related reactions between dinucleotides by a ribozyme. 1987 May 28-Jun 3Nature. 327(6120):343–346. doi: 10.1038/327343a0. [DOI] [PubMed] [Google Scholar]
- Kay P. S., Inoue T. Reactivity of modified ribose moieties of guanosine: new cleavage reactions mediated by the IVS of Tetrahymena precursor rRNA. Nucleic Acids Res. 1987 Feb 25;15(4):1559–1577. doi: 10.1093/nar/15.4.1559. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kay P. S., Menzel P., Inoue T. Two guanosine binding sites exist in group I self-splicing IVS RNAs. EMBO J. 1988 Nov;7(11):3531–3537. doi: 10.1002/j.1460-2075.1988.tb03229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michel F., Hanna M., Green R., Bartel D. P., Szostak J. W. The guanosine binding site of the Tetrahymena ribozyme. Nature. 1989 Nov 23;342(6248):391–395. doi: 10.1038/342391a0. [DOI] [PubMed] [Google Scholar]
- Raines R. T., Sutton E. L., Straus D. R., Gilbert W., Knowles J. R. Reaction energetics of a mutant triosephosphate isomerase in which the active-site glutamate has been changed to aspartate. Biochemistry. 1986 Nov 4;25(22):7142–7154. doi: 10.1021/bi00370a057. [DOI] [PubMed] [Google Scholar]
- Szostak J. W. Enzymatic activity of the conserved core of a group I self-splicing intron. Nature. 1986 Jul 3;322(6074):83–86. doi: 10.1038/322083a0. [DOI] [PubMed] [Google Scholar]
- Tanner N. K., Cech T. R. Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila. Biochemistry. 1987 Jun 16;26(12):3330–3340. doi: 10.1021/bi00386a013. [DOI] [PubMed] [Google Scholar]
- Yarus M. A specific amino acid binding site composed of RNA. Science. 1988 Jun 24;240(4860):1751–1758. doi: 10.1126/science.3381099. [DOI] [PubMed] [Google Scholar]
- Yarus M., Christian E. L. Genetic code origins. Nature. 1989 Nov 23;342(6248):349–350. doi: 10.1038/342349b0. [DOI] [PubMed] [Google Scholar]
- Yarus M., Levine J., Morin G. B., Cech T. R. A Tetrahymena intron nucleotide connected to the GTP/arginine site. Nucleic Acids Res. 1989 Sep 12;17(17):6969–6981. doi: 10.1093/nar/17.17.6969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yarus M. Specificity of arginine binding by the Tetrahymena intron. Biochemistry. 1989 Feb 7;28(3):980–988. doi: 10.1021/bi00429a010. [DOI] [PubMed] [Google Scholar]