Characterization of RNA degradosome from E. coli with mutant polynucleotide phosphorylase

The RNA degradosome is a bacterial protein machine devoted to RNA turnover. Degradosomes and related complexes have been described in Escherichia coli and other prokaryotes as well as in eukaryotes. The integral components of the E. coli RNA degradosome include the endoribonuclease RNase E, the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), the DEAD-box helicase RhlB, and enolase, a glycolytic enzyme commonly implicated in an apparently unrelated process. It is believed that the degradosome coordinates the endo- and exonucleolytic activities of RNase E and PNPase, respectively, whereas the ATP-consuming RhlB helicase would promote the unwinding of double stranded RNA, thus facilitating progression of PNPase through RNA secondary structures. Many questions are still open on the composition, molecular interactions, assembly pathway, mechanism of action, and physiological significance of this molecular machine. To get further insights into the structure and function of this protein complex, we investigated on the properties of RNA degradosomes associated with mutant PNPases with single aminoacid substitutions in different domains and exhibiting different phenotypic traits. The mutant proteins were expressed from plasmid vectors in a PNPase-null E. coli mutant together with a FLAG-tagged RNase E. Degradosomes were then purified by FLAG-affinity chromatography and assayed for the RhlB-promoted degradation of a structured RNA substrate (malE-malF intergenic region) in the presence of ATP (1,2,3). None of mutations tested impaired assembly of PNPase with RNase E and, for most mutants, the RNA-degrading ability of the degradosome correlated with the phosphorolytic activity of the mutant PNPase. An exception was Pnp-E81D, which harbors a single aminoacid substitution on the outer side of the trimerization interface. Although in crude extracts neither phosphorolytic activity nor RNA binding appeared to be impaired, the mutant was defective in autogenous regulation. The RNA degradosome containing Pnp-E81D was unable to degrade the structured RNA substrate. This seemed to correlate with a lower abundance of the RhlB RNA helicase. It thus appears that PNPase participates in the assembly of RhlB in the degradosome and/or in the control of the RNA helicase activity. In addition, these data suggest the participation of the RhlB RNA helicase in PNPase autogenous regulation. References 1. Miczak, A., Kaberdin, V.R., Wei, C.L., and Lin-Chao, S. Proteins associated with RNase E in a multicomponent ribonucleolytic complex. Proc. Natl. Acad. Sci. USA 93 (1996) 3865–3869. 2. Regonesi, M. E., Del Favero, M., Basilico, F., Briani, F., Benazzi, L., Tortora, P., Mauri, P., and Dehò, G. (2006) Analysis of the Escherichia coli RNA degradosome composition by a proteomic approach. Biochimie 88:151-161. 3. Py, B., Higgins, C. F., Krisch, H. M., and Carpousis, A. J. (1996) A DEAD-box RNA helicase in the Escherichia coli RNA degradosome. Nature 381:169-172.