Identification of the Catalytic Subdomain of the VS Ribozyme and Evidence for Remarkable Sequence Tolerance in the Active Site Loop

• Published in: Journal of molecular biology 2002-07, Vol.320 (3), p.443-454
• Main Authors: Sood, Vanita D., Collins, Richard A.
• Format: Article
• Language: English
• Subjects: active site; Base Sequence; Catalytic Domain - genetics; chemical modification; deletion; Endoribonucleases - chemistry; Endoribonucleases - genetics; Endoribonucleases - metabolism; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Neurospora - enzymology; Neurospora - genetics; Nucleic Acid Conformation; ribozyme; RNA, Catalytic - chemistry; RNA, Catalytic - genetics; RNA, Catalytic - metabolism; RNA, Fungal - chemistry; RNA, Fungal - genetics; RNA, Fungal - metabolism; Sequence Deletion; Substrate Specificity
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/S0022-2836(02)00521-1

We show here that the ribozyme domain of the Neurospora VS ribozyme consists of separable upper and lower subdomains. Deletion analysis demonstrates that the entire upper subdomain (helices III/IV/V) is dispensable for site-specific cleavage activity, providing experimental evidence that the active site is contained within the lower subdomain and within the substrate itself. We demonstrate an important role in cleavage activity for a region of helix VI called the 730 loop. Surprisingly, several loop sequences, sizes, and structures at this position can support site-specific cleavage, suggesting that a variety of non-Watson–Crick structures, rather than a specific loop structure, in this region of the ribozyme can contribute to formation of the active site.