The assembly and distribution in vivo of the Escherichia coli RNA degradosome.
We report an analysis in vivo of the RNA degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E-PNPase (polynucleotide phosphorylase), and RNase E-Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present evidence for in vivo proximity of Enolase-PNPase and Enolase-RhlB. The data support a model for the RNA degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.
Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Mexico. Electronic address: ldominguez@cinvestav.mx.
Lilianha
L
Domínguez-Malfavón
León D
LD
Islas
Ben F
BF
Luisi
Refugio
R
García-Villegas
Jaime
J
García-Mena
D053487:DEAD-box RNA Helicases / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060066:Protein Interaction Maps / Q000235:genetics; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; C111244:degradosome; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases
Fluorescence energy transfer; Protein distribution; Protein expression; Protein interaction; RNA degradosome