Assaying DEAD-box RNA helicases and their role in mRNA degradation in Escherichia coli.
The DEAD-box RNA helicases are a ubiquitous family of enzymes involved in processes that include RNA splicing, ribosome biogenesis, and mRNA degradation. In general, these enzymes help to unwind short stretches of double-stranded RNA in processes that involve the remodeling of RNA structure or of ribonucleoprotein complexes. Here we describe work from our laboratory on the characterization of the RhlB of Escherichia coli, a DEAD-box RNA helicase that is part of a multienzyme complex known as the RNA degradosome. RhlB interacts physically and functionally with RNase E and polynucleotide phosphorylase (PNPase), two other components of the RNA degradosome. We describe enzyme assays that demonstrated that the interaction between RhlB and RNase E is necessary for the ATPase and RNA unwinding activities of RhlB. We also describe an mRNA degradation assay that showed that RhlB facilitates the degradation of structured mRNA by PNPase. These assays are discussed in the context of how they have contributed to our understanding of the function of RhlB in mRNA degradation.
Carpousis
Agamemnon J
AJ
Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientifique and Université Paul Sabatier, Toulouse, France.
Khemici
Vanessa
V
Poljak
Leonora
L
D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D011117:Polyribonucleotide Nucleotidyltransferase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D053487:DEAD-box RNA Helicases