Title | Characterization of the NTPase, RNA-binding, and RNA helicase activities of the DEAH-box splicing factor Prp22. |
Publication Type | Journal Article |
Year of Publication | 2005 |
Authors | Tanaka N, Schwer B |
Journal | Biochemistry |
Volume | 44 |
Issue | 28 |
Pagination | 9795-803 |
Date Published | 2005 Jul 19 |
ISSN | 0006-2960 |
Keywords | Adenosine 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 |
Abstract | 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. |
DOI | 10.1021/bi050407m |
Alternate Journal | Biochemistry |
PubMed ID | 16008364 |
Grant List | GM50288 / GM / NIGMS NIH HHS / United States |
Submitted by mam2155 on March 24, 2014 - 4:20pm