Preparation of the Escherichia coli RNase E protein and reconstitution of the RNA degradosome.
The RNA degradosome is a multienzyme complex that plays a key role in the processing of stable RNAs, the degradation of mRNAs, and the action of small regulatory RNAs. Initially discovered in Escherichia coli, similar or related complexes are found in other bacteria. The core of the RNA degradosome is the essential endoribonuclease, RNase E. The C-terminus of this enzyme serves as a scaffold to which other components of the RNA degradosome bind. These ligands include the phosphorolytic 3'-exonuclease, polynucleotide phosphorylase, the DEAD-box RNA helicase, RhlB, and the glycolytic enzyme, enolase. In addition, the DEAD-box RNA helicases CsdA and RhlE and the RNA binding protein, Hfq, may bind to RNase E in place of one or more of the prototypical components. This chapter describes purification of RNase E (the Rne protein), reconstitution of a minimal degradosome that recapitulates the activity of authentic degradosomes, and methods for the assay of the reconstituted complex.
Mackie
George A
GA
Department of Biochemistry Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada
Coburn
Glen A
GA
Miao
Xin
X
Briant
Douglas J
DJ
Prud'homme-Généreux
Annie
A
Stickney
Leigh M
LM
Hankins
Janet S
JS
D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism*
D012313:RNA; D004722:Endoribonucleases; C022537:ribonuclease E