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PMID:19393667

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Citation

Del Campo, M, Mohr, S, Jiang, Y, Jia, H, Jankowsky, E and Lambowitz, AM (2009) Unwinding by local strand separation is critical for the function of DEAD-box proteins as RNA chaperones. J. Mol. Biol. 389:674-93

Abstract

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Delta strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-DeltaP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.

Links

PubMed PMC2769564 Online version:10.1016/j.jmb.2009.04.043

Keywords

Base Sequence; DEAD-box RNA Helicases/genetics; DEAD-box RNA Helicases/metabolism; DNA, Mitochondrial/genetics; DNA, Mitochondrial/metabolism; Genetic Complementation Test; Introns; Molecular Chaperones/genetics; Molecular Chaperones/metabolism; Molecular Sequence Data; Neurospora crassa/genetics; Neurospora crassa/metabolism; Nucleic Acid Conformation; Protein Biosynthesis; RNA/chemistry; RNA/genetics; RNA/metabolism; RNA Splicing; Saccharomyces cerevisiae/genetics; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

ECOLI:SRMB

enables

GO:0003724: RNA helicase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:SRMB

enables

GO:0008186: ATP-dependent activity, acting on RNA

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:SRMB

enables

GO:0004004: ATP-dependent RNA helicase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

ECOLI:SRMB

enables

GO:0016887: ATPase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

Notes

See also

References

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