The paralogous endoribonucleases, RNase E and RNase G, play major roles in intracellular RNA metabolism in Escherichia coli and related organisms. To assay the relative importance of the principal RNA binding sites identified by crystallographic analysis, we introduced mutations into the 5-sensor, the S1 domain, and the Mg ؉2 /Mn ؉2 binding sites. The RNase E/G family of bacterial endoribonucleases is widely distributed among bacteria (1). Both RNase E and RNase G are expressed in Escherichia coli. RNase E was first characterized as an essential processing enzyme required for the maturation of 5 S rRNA 2 (2, 3). It is now known also to be involved in processing the 5Ј-spacer region of 16 S rRNA (4), most tRNA precursors (5, 6), transfer messenger RNA (7), and in the metabolism of many small regulatory RNAs (8, 9). It is also responsible for catalyzing the initial cleavage in the degradation of most mRNAs (10, 11). Furthermore, RNase E is part of a larger complex, the RNA degradosome (12-14). In contrast, RNase G appears to play a more limited role in RNA metabolism. It is responsible for the formation of the mature 5Ј terminus of 16 S rRNA (4, 15) and participates in the degradation of a limited set of mRNAs (16,17). It is not essential, however. Although both enzymes prefer single-stranded substrates, neither displays stringent sequence specificity (18 -20). However, both enzymes are 5Ј-end-dependent; i.e. their activity is stimulated, both in vivo and in vitro by a 5Ј-monophosphorylated terminus on their substrates (21-26). To explain this observation, it was postulated that a 5Ј-phosphate binding pocket exists on the surface of these enzymes (24). This idea has been substantially verified by the crystal structure of the catalytic domain of RNase E in complex with a substrate analog (27). These authors showed that RNase E contains a 5Ј-sensor domain that can interact specifically with a 5Ј-monophosphorylated substrate via contacts with Gly-124, Val-128, Arg-169, and Thr-170 (27).Several investigations have identified potential RNA binding surfaces on RNase E in addition to the 5Ј-sensor, including an arginine-rich region (28 -30) and the S1 domain (31, 32). In addition, the active (catalytic) site itself must contribute to substrate binding. The arginine-rich region, however, lies outside the minimal N-terminal domain of RNase E that is sufficient for enzymatic activity (28 -30). Several residues in the S1 domain could contribute to RNA binding, but only three, Phe-57, Phe-67, and Lys-112 provide obvious contacts to the substrate (27). Thus, it is not clear to what extent the 5Ј-sensor contributes to substrate binding. Indeed, it has been suggested that interaction of RNase E or G with a 5Ј-monophosphorylated substrate increases these enzymes' V max , effectively providing activation of these enzymes (25). Because a crystal structure was not available at the time this work was initiated, we examined instead the role of two types of conserved amino acid residue lying between the S1 domain and residue 400 in RNa...

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 (...

To better understand the roles of the KH and S1 domains in RNA binding and polynucleotide phosphorylase (PNPase) autoregulation, we have identified and investigated key residues in these domains. A convenient pnp::lacZ fusion reporter strain was used to assess autoregulation by mutant PNPase proteins lacking the KH and/or S1 domains or containing point mutations in those domains. Mutant enzymes were purified and studied by using in vitro band shift and phosphorolysis assays to gauge binding and enzymatic activity. We show that reductions in substrate affinity accompany impairment of PNPase autoregulation. A remarkably strong correlation was observed between ␤-galactosidase levels reflecting autoregulation and apparent K D values for the binding of a model RNA substrate. These data show that both the KH and S1 domains of PNPase play critical roles in substrate binding and autoregulation. The findings are discussed in the context of the structure, binding sites, and function of PNPase. Polynucleotide phosphorylase (PNPase) is a conserved, widely distributed phosphorolytic 3=-5= exoribonuclease that may also function under some circumstances as a template-independent RNA polymerase (1; reviewed in reference 2). Although it is not essential, deletions of its gene (pnp) are synthetically lethal when either RNase II or RNase R, both of which are hydrolytic 3=-5= exoribonucleases, is deficient (3-5). Strains deficient in PNPase are also sensitive to cold shock and other stresses (6-9). Thus, PNPase is believed to play significant roles in mRNA turnover and other aspects of RNA processing and metabolism (4, 10-12). Partial or full structures of PNPase from several microorganisms (13-16), as well as structures of the related archaeal exosome (17, 18), have shed considerable light on its mechanism of action. Bacterial PNPase is composed of three identical subunits. Each subunit consists of two tandem globular domains (residues 8 to 210 and 312 to 541; see Fig. 1a) derived from RNase PH that form a core whose central channel is accessible from both the upper and lower surfaces of the core (14,19). Each subunit also contains a C-terminal extension that consists of two additional small domains, KH and S1 (residues 551 to 591 and 622 to 691, respectively; Fig. 1), which are positioned on the upper surface of the core. The KH and S1 domains have been implicated in autoregulation (20), in resistance to cold shock (7), in pathogenesis (21), and in substrate binding (22). Although a solution structure of the S1 domain from Escherichia coli PNPase has been available (13), the positions of the S1 and KH domains relative to the core have been elucidated only recently from the structure of PNPase from Caulobacter crescentus (16). The S1 and KH domains do not appear to be required for the enzymatic activity of PNPase (20,22,23). Nonetheless, deletion of the S1, the KH or both domains results in significant loss of RNA binding and inefficient enzymatic turnover (22).PNPase from E. coli and other organisms exhibits strong autoregulation...

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