Altering the Divalent Metal Ion Preference of RNase E

Abstract

RNase E is a major intracellular endoribonuclease in many bacteria and participates in most aspects of RNA processing and degradation. RNase E requires a divalent metal ion for its activity. We show that only Mg 2؉ RNase E is a 5=-end-dependent endoribonuclease that plays a central role in stable RNA processing and mRNA turnover in Escherichia coli (1). RNase E and its paralog, RNase G, are found in many bacteria but not all (1, 2). In common with many intracellular enzymes of nucleic acid metabolism, RNase E requires a divalent metal ion for activity; in addition, it also requires Zn 2ϩ to stabilize its quaternary structure (3-5). Pioneering work by Misra and Apirion on RNase E demonstrated that the partially purified enzyme requires divalent metal ions with a preference for Mn 2ϩ over Mg 2ϩ (5). With 9S pre-RNA as the substrate, these authors reported optimal concentrations of Mn 2ϩ and Mg 2ϩ as 1 and 5 mM, respectively. Later, Redko et al. used a 3=-fluorescein-labeled decaribonucleotide (BR10F) as the substrate for the purified catalytic domain of RNase E (residues 1 to 529) and reported the optimal Mg 2ϩ concentration as 25 mM (6). Subsequently, the crystal structure of the catalytic domain of RNase E (4) revealed details of how divalent ions contribute to the activity of RNase E. Two conserved residues in the catalytic core, D303 and D346, serve to chelate a Mg 2ϩ ion, while N305 donates an H-bond that helps to anchor D303 (4). The hydration shell surrounding the bound Mg 2ϩ likely serves as the source of a hydroxyl ion that attacks the scissile phosphate (7). In addition, the bound metal ion likely polarizes the phosphate to enhance its reactivity.The ability of RNase E to utilize alternative metal ions in vivo has not been explored. The intracellular concentration of Mg 2ϩ in E. coli can reach almost 200 mM (8, 9); however, most Mg 2ϩ ions are chelated (e.g., by ribosomes [10,11]) and the free concentration is only 1 to 5 mM (8)(9)(10)12). This implies that the activity of RNase E may be limited by the availability of divalent metal ions. However, since RNA binds Mg 2ϩ , the interaction of substrates with RNase E may increase the local concentration of metal ions (7). The concentration of free Mn 2ϩ inside E. coli has not been reported to our knowledge, but total Mn 2ϩ can range from 15 M in cells cultured in unsupplemented defined medium to 150 M in rich medium thanks to active transport mechanisms (13,14). Most intracellular Mn 2ϩ is likely to be chelated resulting in a free pool whose concentration lies in the low micromolar range. Moreover, although Mn 2ϩ is a requirement for pathogenesis in related organisms such as Salmonella enterica serovar Typhimurium (13), and at least 70 enzymes are reported to be able to utilize Mn 2ϩ in E. coli (www.ecocyc.org), relatively few enzymes in E. coli absolutely require Mn 2ϩ . Such enzymes include Mn-dependent superoxide dismutase (encoded by sodA), several glycolytic enzymes and guanosine 3=-diphosphate 5=-triphosphate 3=-diphosphatase encoded by spoT (13).We...

Substrate Recognition and Autoinhibition in the Central Ribonuclease RNase E

“…We first tested binding of the sRNA RprA to RNase E (1–529) WT and the 8x mutant. Using bio-layer interferometry, we investigated the binding with mono- and triphosphorylated sRNA in buffer conditions that do not support cleavage ( Thompson et al., 2015 ) ( Figure 5 A). WT RNase E was capable of binding both 5′ monophosphorylated and 5′ triphosphorylated RprA with similar nanomolar affinity (15.6 ± 4.5 nM and 8.6 ± 7.2 nM, respectively).…”

Bacterial ribonucleases and their roles in RNA metabolism

“…The approximately N-terminal half of the protein contains the RNase E active site and multiple sub-domains including an S1 RNA-binding domain and a 5’ monophosphate sensor domain, among others (McDowall and Cohen 1996; Bycroft et al 1997; Callaghan et al 2005). There are also two aspartic acid residues at positions 303 and 346 required for metal ion coordination at the active site and essential for phosphate backbone cleavage (Thompson et al 2015). The N-terminal portion of RNase E organizes into the catalytically active tetramer in solution dependent on a sub-domain in the 410–510 region of the protein (Callaghan et al 2005).…”

An archaeal RNA binding protein, FAU-1, is a novel ribonuclease related to rRNA stability in Pyrococcus and Thermococcus

“…A divalent metal ion is typically required for RNase activity, and the activity of RNase E is reportedly dependent on Mg 2+ and/or Mn 2+ ions 17 , 19 . A Zn 2+ ion is also required for RNase E to form a tetrameric structure, and consequently indirectly affects its catalytic activity 18 .…”

“…In Escherichia coli , RNase E is involved in the processing of precursor 5S rRNA and 16S rRNA, and is also associated with the stability of mature 16S rRNA and 23S rRNA 9 , 15 , 16 . RNase E requires either Mg 2+ or Mn 2+ ions for its efficient RNase activity and depends on a Zn 2+ ion to form a functional tetramer 3 , 17 – 19 .…”