Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates.
Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ∼70-85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III.
Department of Chemistry, Temple University, Philadelphia, PA, USA.
Zhongjie
Z
Shi
Rhonda H
RH
Nicholson
Ritu
R
Jaggi
Allen W
AW
Nicholson
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D020029:Base Pairing; D001483:Base Sequence; D055162:Biocatalysis; D002413:Cations, Divalent / Q000737:chemistry; D004795:Enzyme Stability; D005973:Glutamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000378:metabolism*; D012492:Salts / Q000737:chemistry; D013696:Temperature
D002413:Cations, Divalent; D012322:RNA Precursors; D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D012492:Salts; D005973:Glutamine; D043244:Ribonuclease III