High-Throughput Genetic Identification of Functionally Important Regions of the Yeast DEAD-Box Protein Mss116p
| Autor: | Mark Del Campo, Rachel Z. Wolf, Kathryn G. Turner, Georg Mohr, Alan M. Lambowitz, Benjamin Gilman |
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| Rok vydání: | 2011 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Protein Conformation RNA Splicing Amino Acid Motifs Immunoblotting Molecular Sequence Data Saccharomyces cerevisiae Crystallography X-Ray Article DEAD-box RNA Helicases Evolution Molecular Structural Biology Amino Acid Sequence Molecular Biology Conserved Sequence Genetics Binding Sites Sequence Homology Amino Acid biology Ribozyme Intron RNA Helicase RNA Fungal Blotting Northern Non-coding RNA RNA Helicase A Cell biology RNA editing Mutation RNA splicing Mutagenesis Site-Directed biology.protein Protein Binding |
| Zdroj: | Journal of Molecular Biology. 413:952-972 |
| ISSN: | 0022-2836 |
| DOI: | 10.1016/j.jmb.2011.09.015 |
| Popis: | The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone that functions in splicing mitochondrial group I and group II introns. Recent X-ray crystal structures of Mss116p in complex with ATP analogs and single-stranded RNA show that the helicase core induces a bend in the bound RNA, as in other DEAD-box proteins, while a C-terminal extension induces a second bend, resulting in RNA crimping. Here, we illuminate these structures by using high-throughput genetic selections, unigenic evolution, and analyses of in vivo splicing activity to comprehensively identify functionally important regions and permissible amino acid substitutions throughout Mss116p. The functionally important regions include those containing conserved sequence motifs involved in ATP and RNA binding or interdomain interactions, as well as previously unidentified regions, including surface loops that may function in protein-protein interactions. The genetic selections recapitulate major features of the conserved helicase motifs seen in other DEAD-box proteins, but also show surprising variations, including multiple novel variants of motif III (SAT). Patterns of amino acid substitutions indicate that the RNA bend induced by the helicase core depends upon ionic and hydrogen-bonding interactions with the bound RNA; identify a subset of critically interacting residues; and indicate that the bend induced by the C-terminal extension results primarily from a steric block. Finally, we identified two conserved regions, one the previously noted post-II region in the helicase core and the other in the C-terminal extension, which may help displace or sequester the opposite RNA strand during RNA unwinding. |
| Databáze: | OpenAIRE |
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