The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E. Using an E. coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E. Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself. However, binding of RhlB or PNPase to enolase was not detected under the same conditions. BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase. Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase. These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.RNA metabolism is a complex process affecting the control of gene expression. In bacteria, a multicomponent ribonucleolytic complex termed the RNA degradosome (1-4) has been identified as playing an important role in the control of mRNA degradation (for recent reviews, see Refs. 5-11). The multicomponent complex consists of: the RNA endonuclease RNase E, whose activity is essential for Escherichia coli cell growth (12-14), RNA processing (15, 16), and degradation (17, 18); the 3Ј-5Ј exoribonuclease PNPase (19); RhlB RNA helicase (20); and enolase (21), an enzyme involved in the glycolytic pathway and other chaperonin proteins (3,22). Interestingly, in addition to mRNAs, highly structured, stable RNA fragments have also been found to be associated with RNA degradosome complexes (3, 23), which implies quality control by the RNA degradosome for the biogenesis of stable RNAs. Degradosome complexity and its cooperation with individual protein components acting on degradation-targeted RNA, in vivo, remains to be discovered.Various approaches revealing protein-protein interactions in the degradosome indicate that the C-terminal region of RNase E serves as a scaffold that directly binds PNPase, RhlB RNA helicase, and enolase (24,25). No other interactions among these component proteins have been detected (25) or reported. Recently, a mini-degradosome complex (26) containing the scaffold region (without the N-terminal enzymatic region of RNase E), RhlB RNA helicase, and PNPase was reconstituted in vitro. These experiments revealed a functional inte...
