Mechanistic characterization of the DEAD-box RNA helicase Ded1 from yeast as revealed by a novel technique using single-molecule magnetic tweezers.
Nucleic Acids Res
; 47(7): 3699-3710, 2019 04 23.
Article
em En
| MEDLINE
| ID: mdl-30993346
ABSTRACT
DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Proteínas de Saccharomyces cerevisiae
/
RNA Helicases DEAD-box
Limite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Ano de publicação:
2019
Tipo de documento:
Article
País de afiliação:
França