Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro

J Mol Biol. 2008 Feb 1;375(5):1344-64. doi: 10.1016/j.jmb.2007.11.041. Epub 2007 Nov 22.


The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended alpha-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted alpha-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the alpha-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphatases / antagonists & inhibitors
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acids, Basic / chemistry
  • Binding Sites
  • Computational Biology / methods
  • Conserved Sequence
  • Crystallography, X-Ray
  • DEAD-box RNA Helicases / chemistry*
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / isolation & purification
  • DEAD-box RNA Helicases / metabolism
  • Drosophila Proteins / chemistry
  • Escherichia coli / genetics
  • Evolution, Molecular
  • Gene Silencing
  • Genetic Complementation Test
  • Hydrophobic and Hydrophilic Interactions
  • In Vitro Techniques
  • Introns
  • Isoelectric Point
  • Kinetics
  • Mitochondria / metabolism
  • Molecular Chaperones / metabolism
  • Molecular Sequence Data
  • Mutation, Missense
  • Plasmids
  • Protein Binding
  • Protein Biosynthesis
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • RNA / metabolism
  • RNA Splicing
  • RNA, Catalytic / metabolism
  • RNA, Fungal / metabolism
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Temperature


  • Amino Acids, Basic
  • Drosophila Proteins
  • Molecular Chaperones
  • RNA, Catalytic
  • RNA, Fungal
  • Saccharomyces cerevisiae Proteins
  • RNA
  • Adenosine Triphosphatases
  • DED1 protein, S cerevisiae
  • MSS116 protein, S cerevisiae
  • vas protein, Drosophila
  • DEAD-box RNA Helicases