Evidence in vivo that the DEAD-box RNA helicase RhlB facilitates the degradation of ribosome-free mRNA by RNase E.
The RNA degradosome of Escherichia coli is a ribonucleolytic multienzyme complex containing RNase E, polynucleotide phosphorylase, RhlB, and enolase. Previous in vitro and in vivo work has shown that RhlB facilitates the exonucleolytic degradation of structured mRNA decay intermediates by polynucleotide phosphorylase in an ATPase-dependent reaction. Here, we show that deleting the gene encoding RhlB stabilizes a lacZ mRNA transcribed by bacteriophage T7 RNA polymerase. Deleting the gene encoding enolase has little if any effect. Other messages transcribed by T7 polymerase are also stabilized by DeltarhlB. The effect of point mutations inactivating RhlB is comparable with the effect of deleting the gene. Primer extension analysis of the lacZ message indicates that RhlB facilitates endoribonucleolytic cleavage by RNase E, demonstrating a functional interaction between the RNA helicase and the endoribonuclease. The possible physiological role of an RhlB-RNase E pathway and the mechanisms by which RhlB could facilitate RNase E cleavage are discussed.
Khemici
Vanessa
V
Laboratoire de Microbiologie et Génétique Moléculaires, Unité Mixte de Recherche 5100, Centre National de la Recherche Scientifique et Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse, France.
Poljak
Leonora
L
Toesca
Isabelle
I
Carpousis
Agamemnon J
AJ
D015153:Blotting, Western; D053487:DEAD-box RNA Helicases; D017931:DNA Primers / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005838:Genotype; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007763:Lac Operon; D008957:Models, Genetic; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D020365:RNA Helicases / Q000737:chemistry* / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism
D017931:DNA Primers; D029968:Escherichia coli Proteins; D012333:RNA, Messenger; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D011117:Polyribonucleotide Nucleotidyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D001616:beta-Galactosidase; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases