Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach.
The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. Several other proteins have been found associated to the core complex. However, it remains unclear in most cases whether such proteins are occasional contaminants or specific components, and which is their function. To facilitate the analysis of the RNA degradosome composition under different physiological and genetic conditions we set up a simplified preparation procedure based on the affinity purification of FLAG epitope-tagged RNase E coupled to Multidimensional Protein Identification Technology (MudPIT) for the rapid and quantitative identification of the different components. By this proteomic approach, we show that the chaperone protein DnaK, previously identified as a "minor component" of the degradosome, associates with abnormal complexes under stressful conditions such as overexpression of RNase E, low temperature, and in the absence of PNPase; however, DnaK does not seem to be essential for RNA degradosome structure nor for its assembly. In addition, we show that normalized score values obtain by MudPIT analysis may be taken as quantitative estimates of the relative protein abundance in different degradosome preparations.
Regonesi
Maria Elena
ME
Dipartimento di Scienze biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.
Del Favero
Marta
M
Basilico
Fabrizio
F
Briani
Federica
F
Benazzi
Louise
L
Tortora
Paolo
P
Mauri
Pierluigi
P
Dehò
Gianni
G
D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046912:Multiprotein Complexes; D010751:Phosphopyruvate Hydratase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000378:metabolism*
D029968:Escherichia coli Proteins; D046912:Multiprotein Complexes; D012313:RNA; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D010751:Phosphopyruvate Hydratase