Insights into Branch Nucleophile Positioning and Activation from an Orthogonal Pre-mRNA Splicing System in Yeast
| Autor: | Duncan J. Smith, Maria M. Konarska, Charles C. Query |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Spliceosome
Saccharomyces cerevisiae Proteins RNA Splicing Molecular Sequence Data Saccharomyces cerevisiae Sequence alignment Computational biology Biology Article 03 medical and health sciences 0302 clinical medicine RNA Small Nuclear RNA Precursors Animals Humans Molecular Biology 030304 developmental biology Genetics 0303 health sciences Base Sequence Intron RNA Cell Biology biology.organism_classification Introns Duplex (building) RNA splicing Spliceosomes Nucleic Acid Conformation Sequence Alignment 030217 neurology & neurosurgery Small nuclear RNA |
| Zdroj: | Molecular Cell. 34:333-343 |
| ISSN: | 1097-2765 |
| Popis: | The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic centre. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide previously unavailable insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research. |
| Databáze: | OpenAIRE |
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