George A. Mackie

Ribonuclease E is a 5'-end-dependent endonuclease

The selective degradation of messenger RNAs enables cells to regulate the levels of particular mRNAs in response to changes in the environment. Ribonuclease (RNase) E (ref. 1), a single-strand-specific endonuclease that is found in a multi-enzyme complex known as the ‘degradosome’, initiates the degradation of many mRNAs in Escherichia coli,,. Its relative lack of sequence specificity and the presence of many potential cleavage sites in mRNA substrates, cannot explain why mRNA decay frequently proceeds in a net 5′-to-3′ direction. I have prepared covalently closed circular derivatives of natural substrates, the rpsT mRNA encoding ribosomal protein S20 (ref. 2) and the 9S precursor to 5S ribosomal RNA,, and find that these derivatives are considerably more resistant to cleavage in vitro by RNase E than are linear molecules. Moreover, antisense oligo-deoxynucleotides complementary to the 5′ end of linear substrates significantly reduce the latters susceptibility to attack by RNase E. Finally, natural substrates with terminal 5′-triphosphate groups are poorly cleaved by RNase E in vitro, whereas 5′ monophosphorylated substrates are strongly preferred (compare with ref. 13). These results show that RNase E has inherent vectorial properties, with its activity depending on the 5′ end of its substrates; this can account for the direction of mRNA decay in E. coli, the phenomenon of ‘all or none’ mRNA decay, and the stabilization provided by 5′ stem–loop structures.

Physical and functional interactions among RNase E, polynucleotide phosphorylase and the cold-shock protein, CsdA: evidence for a ‘cold shock degradosome’

Escherichia coli contains at least five ATP‐dependent DEAD‐box RNA helicases which may play important roles in macromolecular metabolism, especially in translation and mRNA decay. Here we demonstrate that one member of this family, CsdA, whose expression is induced by cold shock, interacts physically and functionally with RNase E. Three independent approaches show that after a shift of cultures to 15°C, CsdA co‐purifies with RNase E and other components of the RNA degradosome. Moreover, functional assays using reconstituted minimal degradosomes prepared from purified components in vitro show that CsdA can fully replace the resident RNA helicase of the RNA degradosome, RhlB. In addition, under these conditions, CsdA displays RNA‐dependent ATPase activity. Taken together, our data are consistent with a model in which CsdA accumulates during the early stages of cold acclimatization and subsequently assembles into degradosomes with RNase E synthesized in cold‐adapted cultures. These findings show that the RNA degradosome is a flexible macromolecular machine capable of adapting to altered environmental conditions.

RNase E: at the interface of bacterial RNA processing and decay

RNase E is an essential endonuclease that is abundant in many bacteria and plays an important part in all aspects of RNA metabolism. It functions as part of a large macromolecular complex known as the RNA degradosome. Recent evidence suggests that this complex associates with the inner membrane of bacteria, an observation that challenges traditional models in which soluble RNases are proposed to randomly interact with RNAs in the cytosol. In this Review, I summarize the major roles of RNase E in RNA processing and decay and discuss the various mechanisms that regulate its activity. I also propose a new model to rationalize the mechanism of RNase E action in the context of its localization in the bacterial cell.

Ectopic RNase E sites promote bypass of 5′‐end‐dependent mRNA decay in Escherichia coli

In Escherichia coli, 5′‐terminal stem–loops form major impediments to mRNA decay, yet conditions that determine their effectiveness or the use of alternative decay pathway(s) are unclear. A synthetic 5′‐terminal hairpin stabilizes the rpsT mRNA sixfold. This stabilization is dependent on efficient translational initiation and ribosome transit through at least two‐thirds of the coding sequence past a major RNase E cleavage site in the rpsT mRNA. Insertion of a 12–15 residue ‘ectopic’ RNase E cleavage site from either the rne leader or 9S pre‐rRNA into the 5′‐non‐coding region of the rpsT mRNA significantly reduces the stabilizing effect of the terminal stem–loop, dependent on RNase E. A similar insertion into the rpsT coding sequence is partially destabilizing. These findings demonstrate that RNase E can bypass an interaction with the 5′‐terminus, and exploit an alternative ‘internal entry’ pathway. We propose a model for degradation of the rpsT mRNA, which explains the hierarchy of protection afforded by different 5′‐termini, the use of internal entry for bypass of barriers to decay, ‘ectopic sites’ and the role of translating ribosomes.

Stabilization of Circular rpsT mRNA Demonstrates the 5′-End Dependence of RNase E Action in Vivo

RNase E is the major intracellular endonuclease in Escherichia coli. Its ability to cleave susceptible substrates in vitro depends on both the cleavage site itself and the availability of an unstructured 5′ terminus. To test whether RNase E activity is 5′-end-dependent in vivo in the presence of all the components of the RNA degradative machinery, a known substrate, the rpsT mRNA, has been embedded in a permuted group I intron to permit its efficient, precise circularization in E. coli. Circular rpsTmRNAs are 4–6-fold more stable in vivo than their linear counterparts. Even partial inactivation of RNase E activity further enhances this stability 6-fold. However, the stabilization of circular rpsT mRNAs depends strongly on their efficient translation. These results show unambiguously the importance of an accessible 5′-end in controlling mRNA stability in vivoand support a two-step (“looping”) model for RNase E action in which the first step is end recognition and the second is actual cleavage.

Overexpression, Purification, and Properties of Escherichia coli Ribonuclease II

Ribonuclease II (RNase II) is a major exonuclease in Escherichia coli that hydrolyzes single-stranded polyribonucleotides processively in the 3′ to 5′ direction. To understand the role of RNase II in the decay of messenger RNA, a strain overexpressing the rnb gene was constructed. Induction resulted in a 300-fold increase in RNase II activity in crude extracts prepared from the overexpressing strain compared to that of a non-overexpressing strain. The recombinant polypeptide (Rnb) was purified to apparent homogeneity in a rapid, simple procedure using conventional chromatographic techniques and/or fast protein liquid chromatography to a final specific activity of 4,100 units/mg. Additionally, a truncated Rnb polypeptide was purified, solubilized, and successfully renatured from inclusion bodies. The recombinant Rnb polypeptide was active against both [3H]poly(A) as well as a novel (synthetic partial duplex) RNA substrate. The data show that the Rnb polypeptide can disengage from its substrate upon stalling at a region of secondary structure and reassociate with a new free 3′-end. The stalled substrate formed by the dissociation event cannot compete for the Rnb polypeptide, demonstrating that duplexed RNAs lacking 10 protruding unpaired nucleotides are not substrates for RNase II. In addition, RNA that has been previously trimmed back to a region of secondary structure with purified Rnb polypeptide is not a substrate for polynucleotide phosphorylase-like activity in crude extracts. The implications for mRNA degradation and the proposed role for RNase II as a repressor of degradation are discussed.

Function of the Conserved S1 and KH Domains in Polynucleotide Phosphorylase

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 multiple-turnover 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 enzymes 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.

Roles of the 5′‐phosphate sensor domain in RNase E

Viable mutations affecting the 5′‐phosphate sensor of RNase E, including R169Q or T170A, become lethal when combined with deletions removing part of the non‐catalytic C‐terminal domain of RNase E. The phosphate sensor is required for efficient autoregulation of RNase E synthesis as RNase E R169Q is strongly overexpressed with accumulation of proteolytic fragments. In addition, mutation of the phosphate sensor stabilizes the rpsT P1 mRNA as much as sixfold and slows the maturation of 16S rRNA. In contrast, the decay of other model mRNAs and the processing of several tRNA precursors are unaffected by mutations in the phosphate sensor. Our data point to the existence of overlapping mechanisms of substrate recognition by RNase E, which lead to a hierarchy of efficiencies with which its RNA targets are attacked.

Substrate Binding and Active Site Residues in RNases E and G: ROLE OF THE 5′-SENSOR*

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 effect of such mutations has been measured by assays of activity on several substrates as well as by an assay of RNA binding. RNase E R169Q and the equivalent mutation in RNase G (R171Q) exhibit the strongest reductions in both activity (the kcat decrease ∼40- to 100-fold) and RNA binding consistent with a key role for the 5′-sensor. Our analysis also supports a model in which the binding of substrate results in an increase in catalytic efficiency. Although the phosphate sensor plays a key role in vitro, it is unexpectedly dispensable in vivo. A strain expressing only RNase E R169Q as the sole source of RNase E activity is viable, exhibits a modest reduction in doubling time and colony size, and accumulates immature 5 S rRNA. Our results point to the importance of alternative RNA binding sites in RNase E and to alternative pathways of RNA recognition.

The quaternary structure of RNase G from Escherichia coli

RNase G is the endoribonuclease responsible for forming the mature 5′ end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N‐terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non‐denaturing purifications for overexpressed RNase G. Using mass spectrometry and N‐terminal sequencing, we unambiguously identified the N‐terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross‐linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross‐linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit–subunit interface.