The RNA degradosome is a multiprotein complex required for the degradation of highly structured RNAs. We have developed a method for reconstituting a minimal degradosome from purified proteins. Our results demonstrate that a degradosome-like complex containing RNase E, PNPase, and RhlB can form spontaneously in vitro in the absence of all other cellular components. Moreover, ATP-dependent degradation of the malEF REP RNA by the reconstituted, minimal degradosome is indistinguishable from that of degradosomes isolated from whole cells. The Rne protein serves as an essential scaffold in the reconstitution process; however, RNase E activity is not required. Rather, Rne coordinates the activation of RhlB dependent on a 3 single-stranded extension on RNA substrates. A model for degradosome-mediated degradation of structured RNA is presented with its implications for mRNA decay in Escherichia coli.

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multipleturnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.The processing and/or degradation of RNAs, including rRNA, tRNA and mRNA, is a critical posttranscriptional regulatory step. In Escherichia coli, several enzymes which participate in RNA processing and degradation are organized into a macromolecular complex, the RNA degradosome (3,18,22). Major components of the degradosome include RNase E, an 5Ј-end-dependent endonuclease, polynucleotide phosphorylase (PNPase), a phosphate-dependent 3Ј exonuclease, RhlB, a DEAD box RNA helicase, and enolase, an abundant glycolytic enzyme (3,5,27). Other proteins associate with the degradosome in apparently substoichiometric quantities, but only RNase E, PNPase, and RhlB are required to reconstitute the activity of the RNA degradosome in vitro (6).Although their activities are quite different, both RNase E and PNPase share a common structural motif, an S1 (or oligonucleotide/oligosaccharide binding fold) domain, as do RNase G and RNase II (2). The relative locations of the S1 domain in these RNases are shown in Fig. 1a. Its location provides no clue to its function(s) in any of these enzymes. S1 domains are also found in a number of unrelated proteins whose principal common feature is interaction with singlestranded nucleic acids (1,20,30). The solution structure of the S1 domain in PNPase has been determined and consists of five antiparallel ␤-strands with surface-exposed hydrophobic and basic residues (2). The structure of the S1 domain of RNase E has also been determined recently and displays a similar overall fold (8,25). Both of the best-characterized mutations in RNase E, rne-1 (G66S) and rne-3071 (...

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