PMID:11888286
| Citation |
Polach, KJ and Uhlenbeck, OC (2002) Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA. Biochemistry 41:3693-702 |
|---|---|
| Abstract |
Unlike most DEAD/H proteins, the purified Escherichia coli protein DbpA demonstrates high specificity for its 23S rRNA substrate in vitro. Here we describe several assays designed to characterize the interaction of DbpA with its RNA and ATP substrates. Electrophoretic mobility shift assays reveal a sub-nanomolar binding affinity for a 153 nucleotide RNA substrate (R153) derived from the 23S rRNA. High affinity RNA binding requires both hairpin 92 and helix 90, as substrates lacking these structures bind DbpA with lower affinity. AMPPNP inhibition assays and ATP/ADP binding assays provide binding constants for ATP and ADP to DbpA with and without RNA substrates. These data have been used to describe a minimal thermodynamic scheme for the binding of the RNA and ATP substrates to DbpA, which reveals cooperative binding between larger RNAs and ATP with cooperative energies of approximately 1.3 kcal mol(-1). This cooperativity is lost upon removal of helix 89 from R153, suggesting this helix is either the preferred target for DbpA's helicase activity or is a necessary structural element for organization of the target site within R153. |
| Links | |
| Keywords |
Adenosine Diphosphate/metabolism; Adenosine Triphosphate/metabolism; Allosteric Regulation; Base Sequence; DEAD-box RNA Helicases; Escherichia coli Proteins; Molecular Sequence Data; Nucleic Acid Conformation; Protein Binding; RNA Helicases/metabolism; RNA, Ribosomal, 23S/chemistry; RNA, Ribosomal, 23S/metabolism; RNA-Binding Proteins |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.