The RNase E of Escherichia coli has at least two binding sites for DEAD-box RNA helicases: functional replacement of RhlB by RhlE.
The non-catalytic region of Escherichia coli RNase E contains a protein scaffold that binds to the other components of the RNA degradosome. Alanine scanning yielded a mutation, R730A, that disrupts the interaction between RNase E and the DEAD-box RNA helicase, RhlB. We show that three other DEAD-box helicases, SrmB, RhlE and CsdA also bind to RNase E in vitro. Their binding differs from that of RhlB because it is not affected by the R730A mutation. Furthermore, the deletion of residues 791-843, which does not affect RhlB binding, disrupts the binding of SrmB, RhlE and CsdA. Therefore, RNase E has at least two RNA helicase binding sites. Reconstitution of a complex containing the protein scaffold of RNase E, PNPase and RhlE shows that RhlE can furnish an ATP-dependent activity that facilitates the degradation of structured RNA by PNPase. Thus, RhlE can replace the function of RhlB in vitro. The results in the accompanying article show that CsdA can also replace RhlB in vitro. Thus, RhlB, RhlE and CsdA are interchangeable in in vitro RNA degradation assays.
Khemici
Vanessa
V
Laboratoire de Microbiologie et Génétique Moléculaires, UMR 5100, Centre National de la Recherche Scientifique (CNRS) et Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France.
Toesca
Isabelle
I
Poljak
Leonora
L
Vanzo
Nathalie F
NF
Carpousis
Agamemnon J
AJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D000251:Adenosine Triphosphatases; C027900:RNA-dependent ATPase; D020365:RNA Helicases