Probing the Mechanisms of DEAD-Box Proteins as General RNA Chaperones: The C-Terminal Domain of CYT-19 Mediates General Recognition of RNA
| Autor: | Pilar Tijerina, Hari Bhaskaran, and Alan M. Lambowitz, Jacob K. Grohman, Mark Del Campo, Rick Russell |
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| Rok vydání: | 2007 |
| Předmět: |
Protozoan Proteins
Biochemistry Article Tetrahymena thermophila DEAD-box RNA Helicases Papain Animals RNA Catalytic Binding site Binding Sites Base Sequence biology GIR1 branching ribozyme C-terminus Ribozyme RNA Cell biology enzymes and coenzymes (carbohydrates) biology.protein Nucleic Acid Conformation Mammalian CPEB3 ribozyme Hairpin ribozyme VS ribozyme Molecular Chaperones |
| Zdroj: | Biochemistry. 46:3013-3022 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity. |
| Databáze: | OpenAIRE |
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