Determination of key residues for catalysis and RNA cleavage specificity: one mutation turns RNase II into a "SUPER-ENZYME".
RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3'-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different single- or double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."
Barbas
Ana
A
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
Matos
Rute G
RG
Amblar
Mónica
M
López-Viñas
Eduardo
E
Gomez-Puertas
Paulino
P
Arraiano
Cecília M
CM
D019943:Amino Acid Substitution / Q000235:genetics; D000596:Amino Acids / Q000378:metabolism*; D055162:Biocatalysis; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D000596:Amino Acids; D050505:Mutant Proteins; D012313:RNA; D004247:DNA; D005095:Exoribonucleases; C022535:exoribonuclease II