An independently folding domain of RNA tertiary structure within the Tetrahymena ribozyme

• Published in: Biochemistry (Easton) Vol. 32; no. 20; pp. 5291 - 5300
• Main Authors: Murphy, Felicia L, Cech, Thomas R
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 25.05.1993
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Conserved Sequence; Edetic Acid; Ferrous Compounds; Fundamental and applied biological sciences. Psychology; Methylation; Molecular Sequence Data; Mutagenesis; Nucleic Acid Conformation; Nucleic acids; RNA - chemistry; RNA Precursors - chemistry; RNA, Catalytic - chemistry; Rna, ribonucleoproteins; Sulfuric Acid Esters; Tetrahymena thermophila; Tetrahymena thermophila - genetics; Extension; Protozoa; Tetrahymena thermophila; Intron; RNA; Domain structure; Ribozyme; Enzyme; Refolding; Ciliata; Endonuclease; Localization
• DOI: 10.1021/bi00071a003
• ISSN: 0006-2960

The Tetrahymena thermophila pre-rRNA contains a 413-nucleotide self-splicing group I intron. This intron has been converted into a sequence-specific endonuclease or ribozyme. A 160-nucleotide portion of the ribozyme consisting of both highly conserved sequence elements (P4 and P6) and nonconserved peripheral extensions (P5abc and P6ab) was synthesized as a separate molecule. Solvent-based Fe(II)-EDTA, a probe that monitors higher-order RNA structure, revealed a protection pattern that was a large subset of that observed in the whole ribozyme. Data from dimethyl sulfate modification and partial digestion with nucleases were also consistent with maintenance of the proper secondary and tertiary structure in the shortened RNA molecule. Thus, this 160-nucleotide molecule (P4-P6 RNA) is an independently folding domain of RNA tertiary structure. A series of mutations and deletions were made within the P4-P6 domain to further dissect its tertiary structure. Fe(II)-EDTA and dimethyl sulfate analysis of these mutants revealed that the domain consists of two substructures, a localized subdomain involving the characteristic adenosine-rich bulge in P5a, and a subdomain-stabilized structure involving long-range interactions. Therefore, like some proteins, the intron RNA is modular, containing a separable domain and subdomain of tertiary structure.