Abstract
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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.