RhlB helicase rather than enolase is the beta-subunit of the Escherichia coli polynucleotide phosphorylase (PNPase)-exoribonucleolytic complex.
Escherichia coli polynucleotide phosphorylase (PNPase), a protein that has both ribonucleolytic and synthetic capabilities, binds, along with the 48-kDa glycolytic enzyme enolase, the 50-kDa DEAD-box protein RhlB helicase and other cellular proteins to the C-terminal "scaffold" region of RNase E to form a complex termed the RNA degradosome. PNPase itself has been reported to exist as a complex (alpha(3)beta(2)) containing trimers of a catalytic subunit (alpha) and dimers of another subunit (beta). The beta-subunit has been believed to be enolase; we report here that it is instead the RhlB helicase. Whereas interaction between PNPase-alpha and enolase was observed in bacteria that synthesize RNase E having a scaffold region, immunoprecipitates from cells expressing PNPase-alpha, RhlB, and enolase from single-copy chromosomal loci, plus a mutant RNase E protein lacking its C-terminal half, showed direct association of PNPase-alpha only with RhlB. Using affinity chromatography, we found that PNPase-alpha and RhlB form a ribonucleolytically active complex corresponding to the mass calculated previously for alpha(3)beta(2) (i.e., 377-380 kDa), whereas no association between PNPase-alpha and enolase was detected. Chromosomal deletion of the eno gene had no effect on the ability of PNPase to degrade either single- or double-stranded RNAs. Collectively, our findings show that direct interaction between PNPase-alpha and RhlB occurs physiologically in the absence of the RNase E C-terminal region, that enolase association with PNPase-alpha is a consequence of the interaction of both proteins with RNase E, and that, contrary to current notions, enolase is not the beta-subunit of E. coli PNPase complex.
Lin
Pei-Hsun
PH
Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.
Lin-Chao
Sue
S
D001483:Base Sequence; D053487:DEAD-box RNA Helicases; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005095:Exoribonucleases / Q000378:metabolism*; D006868:Hydrolysis; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D011117:Polyribonucleotide Nucleotidyltransferase / Q000737:chemistry / Q000378:metabolism*; D012313:RNA; D020365:RNA Helicases / Q000378:metabolism*
D029968:Escherichia coli Proteins; C034477:RNA primers; D012313:RNA; D011117:Polyribonucleotide Nucleotidyltransferase; D005095:Exoribonucleases; C502431:RhlB protein, E coli; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D010751:Phosphopyruvate Hydratase