Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W.
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.
Purusharth
Rajyaguru Ichchhashankar
RI
Institut für Mikrobiologie und Molekularbiologie der Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany.
Klein
Franziska
F
Sulthana
Shaheen
S
Jäger
Stephanie
S
Jagannadham
Medicharla Venkata
MV
Evguenieva-Hackenberg
Elena
E
Ray
Malay Kumar
MK
Klug
Gabriele
G
D000255:Adenosine Triphosphate / Q000737:chemistry; D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D005095:Exoribonucleases / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D047468:Immunoprecipitation; D011485:Protein Binding; D044224:Pseudomonas syringae / Q000201:enzymology*; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry; D013997:Time Factors; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D012313:RNA; D000255:Adenosine Triphosphate; D004722:Endoribonucleases; D005095:Exoribonucleases; D012260:Ribonucleases; C080868:ribonuclease R; C022537:ribonuclease E; D020365:RNA Helicases; D005990:Glycerol