Linkage of catalysis and 5′ end recognition in ribonuclease RNase J

Abstract

In diverse bacterial species, the turnover and processing of many RNAs is mediated by the ribonuclease RNase J, a member of the widely occurring metallo-β-lactamase enzyme family. We present crystal structures of Streptomyces coelicolor RNase J with bound RNA in pre- and post-cleavage states, at 2.27 Å and 2.80 Å resolution, respectively. These structures reveal snapshots of the enzyme cleaving substrate directionally and sequentially from the 5′ terminus. In the pre-cleavage state, a water molecule is coordinated to a zinc ion pair in the active site but is imperfectly oriented to launch a nucleophilic attack on the phosphate backbone. A conformational switch is envisaged that enables the in-line positioning of the attacking water and may be facilitated by magnesium ions. Adjacent to the scissile bond, four bases are stacked in a tightly sandwiching pocket, and mutagenesis results indicate that this organization helps to drive processive exo-ribonucleolytic cleavage. Like its numerous homologues, S. coelicolor RNase J can also cleave some RNA internally, and the structural data suggest how the preference for exo- versus endo-cleavage mode is linked with recognition of the chemical status of the substrate's 5′ end.

New molecular insights into an archaeal RNase J reveal a conserved processive exoribonucleolysis mechanism of the RNase J family

“…Similar sandwich pockets that sequester bases +2 to +4 are also observed in the RNA‐bound structures of dra‐ and sco‐RNase Js (Supporting Information Fig. S10) (Zhao et al ., ; Pei et al ., ), implying the importance of this sandwich pocket in enzymatic activity. However, only two bases are sequestered in the equivalent Arg266/Phe43 pocket in the structure of tth‐RNase J–2′‐O‐methyl modified RNA (Supporting Information Fig.…”

“…Similar to other RNase J homologs, the recombinant mpy-RNase J displays 5 0 -monophosphate preferential 5 0 -3 0 processive exoribonucleolytic activity (Supporting Information Figs S2 and S3, detailed results presented in Supporting Information). However, distinct from bacterial RNase Js (de la Sierra-Gallay et al, 2008;Pei et al, 2015), and similar to other archaeal RNase J (Clouet-d'Orval et al, 2010), mpy-RNase J is capable of exonucleolytically degrading 5 0 -triphosphorylated RNA completely to the 3 0 end (Supporting Information Fig. S2C and D), but at about a nine fold lower rate than for 5 0 -monophosphorylated RNA (Supporting Information Fig.…”

“…S8B). A similar RNA binding path is predicted for the bacterial tth‐RNase J in endoribonucleolytic mode (Dorleans et al ., ), whereas the 5′‐end sensing for 5′ppp is predicted to trigger an exo‐ to endonucleolytic switch in sco‐RNase J (Pei et al ., ), but not for accommodating 5′ ppp (de la Sierra‐Gallay et al ., ; Pei et al ., ). However, it was recently found that S. aureus RNase J1 can exoribonucleolytically attack the 5′ppp RNA from the 5′ppp end (Taverniti et al ., ; Hausmann et al ., ), and M. smegmatis and B. subtilis RNase J1s also show weak cleavages at the 5′ppp end of RNA (Taverniti et al ., ).…”

“…The RNA translocation mechanism is conserved in the RNase J family Because mutants R267A and F52A abolish exoribonucleolysis, Pei et al predicted that the sandwich pocket assists in driving processive exoribonucleolytic cleavage (Pei et al, 2015). To compare the RNA translocation mechanism employed by archaeal and bacterial RNase Js, we determined the key residues in the 5'p and sandwich pockets of the bacterial sco-RNase J in contribution to RNA translocation in parallel.…”

A label-free aptamer-based biosensor for microRNA detection by the RNA-regulated fluorescence of malachite green

“…[19][20][21] Due to the excellent characteristics of aptamers, many research elds about aptamer-based sensors have developed rapidly. 19,20 The most design concepts of the aptamer-based sensors are to convert binding events between the aptamers and ligands into detectable signal changes, such as colorimetric, 23 electrochemical, 24 chemiluminescent, 25 uorescent, 26,27 and phase changes. 28 Among these aptamerbased sensors, uorescent sensors have attracted more attention in biosensing because of their high sensitivity and the feasibility of quantication.…”