Structural insights into catalysis and dimerization enhanced exonuclease activity of RNase J

Abstract

RNase J is a conserved ribonuclease that belongs to the β-CASP family of nucleases. It possesses both endo- and exo-ribonuclease activities, which play a key role in pre-rRNA maturation and mRNA decay. Here we report high-resolution crystal structures of Deinococcus radiodurans RNase J complexed with RNA or uridine 5′-monophosphate in the presence of manganese ions. Biochemical and structural studies revealed that RNase J uses zinc ions for two-metal-ion catalysis. One residue conserved among RNase J orthologues (motif B) forms specific electrostatic interactions with the scissile phosphate of the RNA that is critical for the catalysis and product stabilization. The additional manganese ion, which is coordinated by conserved residues at the dimer interface, is critical for RNase J dimerization and exonuclease activity. The structures may also shed light on the mechanism of RNase J exo- and endonucleolytic activity switch.

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

“…A and B). This is similar to the corresponding mutants His377A, S379A and H377A/S379A of dra‐RNase J (Zhao et al ., ). Consistently, the double mutation of H384A/S386A remarkably reduced the RNA binding by 5‐fold, while single mutation of His384 or Ser386 had less than twofold reduction (Fig.…”

“…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.…”

“…The crystal structures of RNase J from Thermus thermophilus (tth‐RNase J) and Bacillus subtilis (bsu‐RNase J1) show a two‐zinc ion catalytic center and an RNA binding channel positioned at the interface of the β‐CASP and metallo‐β‐lactamase domains (de la Sierra‐Gallay et al ., ; Dorleans et al ., ; Newman et al ., ). Two recently reported RNA‐bound structures of RNase J from Deinococcus radiodurans (dra‐RNase J) and Streptomyces coelicolor (sco‐RNase J) have elucidated the detailed two‐zinc‐ion catalytic mechanism, in which a water molecule is coordinated by two zinc ions and is activated to be a hydroxyl ion so as to achieve an in‐line nucleophilic attack for hydrolytic cleavage (Zhao et al ., ; Pei et al ., ). As a highly processive exoribonuclease (Mathy et al ., ; Clouet‐d'Orval et al ., ), RNase J has to translocate the ( n –1) RNA products one nucleotide forward to the catalytic site following each 5′‐terminal cleavage.…”

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

“…The model was calculated using molecular dynamics simulation. ( B ) Detailed proposed catalytic mechanism of RNase J based on the crystal structure of the S. coelicolor RNase J/RNA complex and the suggested mechanism for RNase Z of de la Sierra-Gallay et al ( 9 ) and for D. radiodurans RNase J of Zhao et al ( 7 ). The 5′ terminal phosphate may assist in catalysis by orienting a water chain for proton channeling.…”

Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer

“…The predominant 3′-to-5′ exonuclease activity observed for the P207E mutant (Figure 5) provides further evidence for a possible endo-to-exonuclease functional switch controlled by conformational alternations between dimer and monomer. A previous study on RNase J reveals a similar endonuclease-to-exonuclease switch upon monomer-to-dimer conformational change (27). Different from EndoG whose dimer-to-monomer conformational change was induced by protein oxidation, the monomer-to-dimer switch of RNase J is induced by divalent cations, which further stimulate the 5′-to-3′ exonuclease activity of RNase J.…”