Abstract
In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.
Full text
PDF






Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Beaudry A. A., Joyce G. F. Directed evolution of an RNA enzyme. Science. 1992 Jul 31;257(5070):635–641. doi: 10.1126/science.1496376. [DOI] [PubMed] [Google Scholar]
- Berzal-Herranz A., Joseph S., Burke J. M. In vitro selection of active hairpin ribozymes by sequential RNA-catalyzed cleavage and ligation reactions. Genes Dev. 1992 Jan;6(1):129–134. doi: 10.1101/gad.6.1.129. [DOI] [PubMed] [Google Scholar]
- Brown J. W., Haas E. S., James B. D., Hunt D. A., Liu J. S., Pace N. R. Phylogenetic analysis and evolution of RNase P RNA in proteobacteria. J Bacteriol. 1991 Jun;173(12):3855–3863. doi: 10.1128/jb.173.12.3855-3863.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Burke J. M., Berzal-Herranz A. In vitro selection and evolution of RNA: applications for catalytic RNA, molecular recognition, and drug discovery. FASEB J. 1993 Jan;7(1):106–112. doi: 10.1096/fasebj.7.1.8422956. [DOI] [PubMed] [Google Scholar]
- Chowrira B. M., Berzal-Herranz A., Burke J. M. Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme. Biochemistry. 1993 Feb 2;32(4):1088–1095. doi: 10.1021/bi00055a014. [DOI] [PubMed] [Google Scholar]
- Chowrira B. M., Berzal-Herranz A., Burke J. M. Novel guanosine requirement for catalysis by the hairpin ribozyme. Nature. 1991 Nov 28;354(6351):320–322. doi: 10.1038/354320a0. [DOI] [PubMed] [Google Scholar]
- Chowrira B. M., Burke J. M. Binding and cleavage of nucleic acids by the "hairpin" ribozyme. Biochemistry. 1991 Sep 3;30(35):8518–8522. doi: 10.1021/bi00099a003. [DOI] [PubMed] [Google Scholar]
- Chowrira B. M., Burke J. M. Extensive phosphorothioate substitution yields highly active and nuclease-resistant hairpin ribozymes. Nucleic Acids Res. 1992 Jun 11;20(11):2835–2840. doi: 10.1093/nar/20.11.2835. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feldstein P. A., Buzayan J. M., Bruening G. Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA. Gene. 1989 Oct 15;82(1):53–61. doi: 10.1016/0378-1119(89)90029-2. [DOI] [PubMed] [Google Scholar]
- Feldstein P. A., Buzayan J. M., van Tol H., deBear J., Gough G. R., Gilham P. T., Bruening G. Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2623–2627. doi: 10.1073/pnas.87.7.2623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green R., Ellington A. D., Szostak J. W. In vitro genetic analysis of the Tetrahymena self-splicing intron. Nature. 1990 Sep 27;347(6291):406–408. doi: 10.1038/347406a0. [DOI] [PubMed] [Google Scholar]
- Hampel A., Tritz R., Hicks M., Cruz P. 'Hairpin' catalytic RNA model: evidence for helices and sequence requirement for substrate RNA. Nucleic Acids Res. 1990 Jan 25;18(2):299–304. doi: 10.1093/nar/18.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hampel A., Tritz R. RNA catalytic properties of the minimum (-)sTRSV sequence. Biochemistry. 1989 Jun 13;28(12):4929–4933. doi: 10.1021/bi00438a002. [DOI] [PubMed] [Google Scholar]
- Haseloff J., Gerlach W. L. Sequences required for self-catalysed cleavage of the satellite RNA of tobacco ringspot virus. Gene. 1989 Oct 15;82(1):43–52. doi: 10.1016/0378-1119(89)90028-0. [DOI] [PubMed] [Google Scholar]
- Joseph S., Berzal-Herranz A., Chowrira B. M., Butcher S. E., Burke J. M. Substrate selection rules for the hairpin ribozyme determined by in vitro selection, mutation, and analysis of mismatched substrates. Genes Dev. 1993 Jan;7(1):130–138. doi: 10.1101/gad.7.1.130. [DOI] [PubMed] [Google Scholar]
- Levitt M. Detailed molecular model for transfer ribonucleic acid. Nature. 1969 Nov 22;224(5221):759–763. doi: 10.1038/224759a0. [DOI] [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]
- Michel F., Jaeger L., Westhof E., Kuras R., Tihy F., Xu M. Q., Shub D. A. Activation of the catalytic core of a group I intron by a remote 3' splice junction. Genes Dev. 1992 Aug;6(8):1373–1385. doi: 10.1101/gad.6.8.1373. [DOI] [PubMed] [Google Scholar]
- Michel F., Westhof E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol. 1990 Dec 5;216(3):585–610. doi: 10.1016/0022-2836(90)90386-Z. [DOI] [PubMed] [Google Scholar]
- Noller H. F., Woese C. R. Secondary structure of 16S ribosomal RNA. Science. 1981 Apr 24;212(4493):403–411. doi: 10.1126/science.6163215. [DOI] [PubMed] [Google Scholar]
- Pan T., Uhlenbeck O. C. In vitro selection of RNAs that undergo autolytic cleavage with Pb2+. Biochemistry. 1992 Apr 28;31(16):3887–3895. doi: 10.1021/bi00131a001. [DOI] [PubMed] [Google Scholar]
- Pyle A. M., Murphy F. L., Cech T. R. RNA substrate binding site in the catalytic core of the Tetrahymena ribozyme. Nature. 1992 Jul 9;358(6382):123–128. doi: 10.1038/358123a0. [DOI] [PubMed] [Google Scholar]
- Robertson D. L., Joyce G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature. 1990 Mar 29;344(6265):467–468. doi: 10.1038/344467a0. [DOI] [PubMed] [Google Scholar]
- Rubino L., Tousignant M. E., Steger G., Kaper J. M. Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus. J Gen Virol. 1990 Sep;71(Pt 9):1897–1903. doi: 10.1099/0022-1317-71-9-1897. [DOI] [PubMed] [Google Scholar]
- Scaringe S. A., Francklyn C., Usman N. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 1990 Sep 25;18(18):5433–5441. doi: 10.1093/nar/18.18.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Szostak J. W. In vitro genetics. Trends Biochem Sci. 1992 Mar;17(3):89–93. doi: 10.1016/0968-0004(92)90242-2. [DOI] [PubMed] [Google Scholar]
- Williamson C. L., Tierney W. M., Kerker B. J., Burke J. M. Site-directed mutagenesis of core sequence elements 9R', 9L, 9R, and 2 in self-splicing Tetrahymena pre-rRNA. J Biol Chem. 1987 Oct 25;262(30):14672–14682. [PubMed] [Google Scholar]