Cooperative binding of ATP and RNA substrates to the DEAD/H protein DbpA.
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.
Polach
Kevin J
KJ
Department of Chemistry and Biochemistry, UCB 215, University of Colorado, Boulder, Colorado 80309-0215, USA.
Uhlenbeck
Olke C
OC
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000494:Allosteric Regulation; D001483:Base Sequence; D053487:DEAD-box RNA Helicases; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020365:RNA Helicases / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins
D029968:Escherichia coli Proteins; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C065908:dbpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases