Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

Abstract

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 (...

The evolutionarily conserved subunits Rrp4 and Csl4 confer different substrate specificities to the archaeal exosome

“…The data suggest that the 25 nt poly(A), too short to bind simultaneously to the central channel of the hexamer and to certain Rrp4 domain(s), is displaced by a next substrate, even if this substrate is shorter. Our observation is in agreement with the two-step model of substrate recognition by E. coli PNPase, and by the proposed key role of its S1 and KH domains for displacement of a stalled substrate and for substrate release [21]. According to this model, a stalled poly(A) degradation product of 25 nt (previously described in Ref.…”

The Pyrococcus Exosome Complex

“…9). In our model, the 3Ј-end of RNA reaches the catalytic centers through the central channel that can accommodate a single-stranded RNA molecule (10,33). As predicted by the crystal structure of S. solfataricus exosome core (14), the aRrp4 protein may be located at the hydrophobic patches in the opposite side of the ring.…”

“…This result is similar to that observed during the preparation of the T (primer-dependent) form of PNPase from the I (primer-independent) form by limited tryptic digestion (36). During the preparation of this manuscript, Stickney et al (33) clearly showed that limited proteolytic digestion removes the RNA binding domains from E. coli PNPase (33), confirming the susceptibility of this region to proteolysis. In that work it is also proposed that the RNA binding domains of PNPase are in an extended conformation (33).…”

“…As predicted by the crystal structure of S. solfataricus exosome core (14), the aRrp4 protein may be located at the hydrophobic patches in the opposite side of the ring. The aRrp4 function is to bring the RNA substrate to the catalytic centers in a way similar to the recently proposed model for bacterial PNPase (33) and then regulate the exosome activity. Although the SAXS models indicate an extended conformation of aRrp4 in solution, it is possible that this protein may adopt a more globular shape in some conditions (as was observed in gel filtration experiments).…”

“…RNA polymerization was detected in greater than 0.5-pmol concentrations of those complexes. This may be the first evidence of regulation of the exosome activity by an RNA-binding protein and would be similar to the RNA phosphorylase activity of full-length and RNA binding deletion mutant of bacterial PNPase (33). Detailed biochemical analyses are still necessary to understand the influence of RNA binding properties of aRrp4 in the RNase activity or substrate selection by the complex.…”