Interaction of Escherichia coli DbpA with 23S rRNA in different functional states of the enzyme.
DEx(D)/(H) proteins catalyze structural rearrangements in RNA by coupling ATP hydrolysis to the destabilization of RNA helices or RNP complexes. The Escherichia coli DEx(D)/(H) protein DbpA specifically recognizes a region within the catalytic core of 23S rRNA. To better characterize the interaction of DbpA with this region and to identify changes in the complex between different nucleotide-bound states of the enzyme, RNase T1, RNase T2, kethoxal and DMS footprinting of DbpA on a 172 nt fragment of 23S rRNA were performed. A number of protections identified in helices 90 and 92 were consistent with biochemical experiments measuring the RNA binding and ATPase activity of DbpA with truncated RNAs. When DbpA was bound with AMPPNP, but not ADP, several additional footprints were detected in helix 93 and the single-stranded region 5' of helix 90, suggesting binding of the helicase domains of DbpA at these sites. These results propose that DbpA can act at multiple sites and hint at the targets of its biological activity on rRNA.
Karginov
Fedor V
FV
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
Uhlenbeck
Olke C
OC
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D053487:DEAD-box RNA Helicases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D023282:Nuclease Protection Assays; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism
D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D016601:RNA-Binding Proteins; D012260:Ribonucleases; D000251:Adenosine Triphosphatases; C065908:dbpA protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases