Structural highlights
4w7s is a 2 chain structure with sequence from Saccharomyces cerevisiae S288C. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
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| Method: | X-ray diffraction, Resolution 2.542Å |
| Ligands: | , , , , |
| Resources: | FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT |
Function
PRP28_YEAST ATP-dependent RNA helicase involved in mRNA splicing. May destabilize the U1/5'-splice site duplex to permit an effective competition for the 5'-splice site by the U6 snRNA, resulting in the switch between U1 and U6 at the 5'-splice site. May also act to unwind the U4/U6 base-pairing interaction in the U4/U6/U5 snRNP, facilitating the first covalent step of splicing.[1] [2] [3]
Publication Abstract from PubMed
Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1-89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127-588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127-588) comprises two RecA-like domains splayed widely apart. AMPPNP*Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP*Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28*AMPPNP structure, and that of the Drosophila Vasa*AMPPNP*Mg2+*RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.
Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28.,Jacewicz A, Schwer B, Smith P, Shuman S Nucleic Acids Res. 2014 Oct 10. pii: gku930. PMID:25303995[4]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Staley JP, Guthrie C. An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. Mol Cell. 1999 Jan;3(1):55-64. PMID:10024879
- ↑ Chen JY, Stands L, Staley JP, Jackups RR Jr, Latus LJ, Chang TH. Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol Cell. 2001 Jan;7(1):227-32. PMID:11172727
- ↑ Strauss EJ, Guthrie C. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 1991 Apr;5(4):629-41. PMID:2010088
- ↑ Jacewicz A, Schwer B, Smith P, Shuman S. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28. Nucleic Acids Res. 2014 Oct 10. pii: gku930. PMID:25303995 doi:http://dx.doi.org/10.1093/nar/gku930
