Abstract
Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.
Highlights
RNA maturation and degradation involve the action of different ribonucleases, some of which are organized in protein complexes
P. syringae RNase R Is in a Complex with RNase E and RhlE—To enrich P. syringae RNase E with associated proteins, we used the procedure already successfully applied for isolation of the RNase E-based protein complexes of E. coli and R. capsulatus
The 118-kDa RNase E of R. capsulatus and E. coli migrates in SDS-PAGE like a 180-kDa protein [1, 13]
Summary
RNA maturation and degradation involve the action of different ribonucleases, some of which are organized in protein complexes. Escherichia coli possesses an RNA-degrading complex called degradosome It is organized by the endoribonuclease RNase E, which is tightly associated with the exoribonuclease polynucleotide phosphorylase (PNPase), the DEAD-box helicase RhlB, and enolase [1,2,3]. Not all bacteria have a gene for RNase E (rne), and some bacteria possess only short RNase E homologues without potential protein binding domains [12] They most probably do not harbor degradosomes. The available data imply that the bacterial degradosome consists of RNase E, DEAD-box helicase(s), the phosphate-dependent exoribonuclease PNPase, and additional subunits, which vary in the different species. We found that RNase E of this strain interacts with the hydrolytic exoribonuclease RNase R in a protein complex Another component of the complex is the DEAD-box helicase RhlE. Our data reveal the existence of a bacterial RNA-degrading complex of unexpected structure
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