Action of RNase II and Polynucleotide Phosphorylase against RNAs Containing Stem-Loops of Defined Structure

Abstract

The 335 exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3 extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.In the bacterium Escherichia coli, degradation of mRNA is almost always initiated by an endoribonuclease, usually RNase E (1, 26). At least two 3Ј exoribonucleases subsequently attack the newly created 3Ј termini generated by RNase E. One of these, RNase II, a monomer with a molecular mass of 72.5 kDa, is hydrolytic (31) and accounts for up to 90% of the exoribonucleolytic activity in crude extracts (12). The other, polynucleotide phosphorylase (PNPase), a trimer with 78-kDa subunits, is phosphorolytic and accounts for the remaining 10% of the exoribonuclease activity in E. coli extracts (12). Although strains singly mutated in the genes encoding RNase II (rnb) or PNPase (pnp) exhibit a mild phenotype, double mutants deficient in both PNPase and RNase II are inviable (13). This finding has been interpreted to indicate that these exonucleases are functionally redundant but collectively essential. Other data, however, suggest that RNase II and PNPase are not functionally equivalent but are differentially sensitive to RNA secondary structure (3,7,8,16,19,24,28). In such cases, PNPase is required for the degradation of highly structured RNAs and RNase II cannot substitute (7,8,19). Moreover, RNase II, but not PNPase, may actually stabilize some RNAs (3,28). In addition, while RNase II behaves as a soluble monomeric enzyme (31), PNPase can be assembled into a multienzyme complex, the degradosome (5, 27, 29). In the complex, PNPase can degrade extensively structured RNA substrates in concert with RhlB, a putative DEAD-box RNA helicase (10, 29). Alternatively, the action of PNPase against folded RNAs can be stimulated by prior 3Ј polyadenylation of such substrates (2, 33). RNase II can also be stimulated by polyadenylation in vitro, but to a more limited extent (7).In order to resolve the paradoxical properties of RNase II and PNPase, we compared their abilities to degrade short synthetic RNA substrates containing a single stem-loop of defined size and thermal stability. This would permit us to test directly whether both enzymes are functionally equivalent. In addition, we could measure the minimum size of base-paired stems which would stall each enzyme and consequently pred...

Direct observation of processive exoribonuclease motion using optical tweezers

“…Therefore, given the absence of a separate source of energy, the processive unwinding of RNA must be driven by the degradation of the RNA substrate. Nevertheless, the application of an external load is expected to perturb the energy landscape for the unwinding reaction, favoring the melting of any double-stranded regions and thereby influencing the velocity and processivity of PNPase and RNase R. This effect may explain why we observed greater processivity for PNPase than suggested by a previous estimate (18). Because the external load primarily affects the melting of the duplex, RNase R and PNPase are expected to move at least as fast and as far on ssRNA where no additional energy is required for unwinding.…”

“…These domains are thought to bind RNA upstream of the catalytic sites, and recent structural evidence suggests that they may guide the substrate into the central channel through a "hands gripping a rope" mechanism (16,17). By itself, PNPase may stall on structured RNA, particularly on G:C hairpins comprising more than six base pairs (18). PNPase often associates with the DEAD-box helicase RhlB to move through structured regions and generally does so in the context of the multienzyme degradosome (2,19).…”

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

“…rHCF107 blocked PNPase-mediated RNA degradation ∼14 nt downstream of the minimal HCF107 binding site, whereas it blocked RNase R at two positions, mapping 2 and 6 nt downstream of the minimal HCF107 binding site. This difference in RNase R and PNPase behavior likely reflects a difference in the structure of these enzymes: PNPase stalls ∼9 nt downstream from the base of stable RNA hairpins because of steric interference with the enzyme's narrow neck (26,27). Thus, the positions at which rHCF107 blocks PNPase and RNase R correlate well with the 3′ boundary of HCF107's binding site.…”

Polynucleotide phosphorylase is a global regulator of virulence and persistency in Salmonella enterica

“…The exoribonuclease activity of PNPase is known to halt stable stem-loop structures (49), whereas the DNA contained in SPI 1 BALB/c mice (female, 6 -8 weeks of age) were challenged orally (14) with a dose corresponding to LD 30. Mice surviving the acute phase of the infection (7-20 days postinfection) were killed 28 -30 days postinfection, and their spleens were removed for enumeration of bacteria. *Persistent infection was defined by the presence of splenic bacteria and splenomegaly (mean spleen weight 0.97 Ϯ 0.34 g, as compared to 0.16 Ϯ 0.05 g for sterile spleens recovered from the survivors).…”