Nonspecific binding to structured RNA and preferential unwinding of an exposed helix by the CYT-19 protein, a DEAD-box RNA chaperone.
We explore the interactions of CYT-19, a DExD/H-box protein that functions in folding of group I RNAs, with a well characterized misfolded species of the Tetrahymena ribozyme. Consistent with its function, CYT-19 accelerates refolding of the misfolded RNA to its native state. Unexpectedly, CYT-19 performs another reaction much more efficiently; it unwinds the 6-bp P1 duplex formed between the ribozyme and its oligonucleotide substrate. Furthermore, CYT-19 performs this reaction 50-fold more efficiently than it unwinds the same duplex free in solution, suggesting that it forms additional interactions with the ribozyme, most likely using a distinct RNA binding site from the one responsible for unwinding. This site can apparently bind double-stranded RNA, as attachment of a simple duplex adjacent to P1 recapitulates much of the activation provided by the ribozyme. Unwinding the native P1 duplex does not accelerate refolding of the misfolded ribozyme, implying that CYT-19 can disrupt multiple contacts on the RNA, consistent with its function in folding of multiple RNAs. Further experiments showed that the P1 duplex unwinding activity is virtually the same whether the ribozyme is misfolded or native but is abrogated by formation of tertiary contacts between the P1 duplex and the body of the ribozyme. Together these results suggest a mechanism for CYT-19 and other general DExD/H-box RNA chaperones in which the proteins bind to structured RNAs and efficiently unwind loosely associated duplexes, which biases the proteins to disrupt nonnative base pairs and gives the liberated strands an opportunity to refold.
Tijerina
Pilar
P
Department of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Bhaskaran
Hari
H
Russell
Rick
R
D000818:Animals; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000378:metabolism; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000235:genetics
D018832:Molecular Chaperones; D016337:RNA, Catalytic; D016053:RNA, Protozoan; D053487:DEAD-box RNA Helicases