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Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22.

TitleCharacterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22.
Publication TypeJournal Article
Year of Publication2005
AuthorsTanaka N, Schwer B
Date Published2005 Jul 19
KeywordsAdenosine Triphosphatases, Adenosine Triphosphate, Binding Sites, Cytidine Triphosphate, DEAD-box RNA Helicases, DNA Helicases, Guanosine Triphosphate, Hydrolysis, Nucleic Acid Heteroduplexes, Nucleoside-Triphosphatase, Poly A, RNA Helicases, RNA Splicing, RNA, Fungal, Saccharomyces cerevisiae Proteins, Spliceosomes, Substrate Specificity, Uridine Triphosphate

The DEAH protein Prp22 is important for the second transesterification step of pre-mRNA splicing, and it is essential for releasing mature mRNA from the spliceosome. Recombinant Prp22 has RNA-stimulated ATPase and ATP-dependent unwinding activities, which are crucial for the mRNA release step. In this study, we characterize the RNA-binding, NTP hydrolysis, and RNA unwinding functions of Prp22. Using nitrocellulose filter binding assays, we determined that the apparent affinity of Prp22 is approximately 20-fold greater for single-stranded RNA than for single-stranded DNA or duplex nucleic acids. Inclusion of hydrolyzable ATP in binding reactions increased the apparent K(D) for RNA by 3-4-fold. The Prp22-RNA interaction is influenced by the length of the RNA chain, and the apparent K(D) values for poly(A)(40) and poly(A)(10) are 17 and 140 nM, respectively. RNA-stimulated ATP hydrolysis is similarly affected by chain length, and optimal activity requires RNA oligomers of >or=20 nt. We show that Prp22 can hydrolyze all common NTPs and dNTPs with comparable efficiencies and that Prp22 unwinds RNA duplexes with 3' to 5' directionality.

Alternate JournalBiochemistry
PubMed ID16008364
Grant ListGM50288 / GM / NIGMS NIH HHS / United States

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