Characterization of the functional domains of Escherichia coli RNase II.
RNase II is a single-stranded-specific 3'-exoribonuclease that degrades RNA generating 5'-mononucleotides. This enzyme is the prototype of an ubiquitous family of enzymes that are crucial in RNA metabolism and share a similar domain organization. By sequence prediction, three different domains have been assigned to the Escherichia coli RNase II: two RNA-binding domains at each end of the protein (CSD and S1), and a central RNB catalytic domain. In this work we have performed a functional characterization of these domains in order to address their role in the activity of RNase II. We have constructed a large set of RNase II truncated proteins and compared them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants were determined using different single- or double-stranded substrates. The results obtained revealed that S1 is the most important domain in the establishment of stable RNA-protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. In addition, we also demonstrate that the N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. Moreover, the biochemical results obtained with RNB mutant that lacks both putative RNA-binding domains, revealed the presence of an additional region involved in RNA binding. Such region, was identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain.
Amblar
Mónica
M
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal.
Barbas
Ana
A
Fialho
Arsénio M
AM
Arraiano
Cecília M
CM
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid
D012329:RNA, Bacterial; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D005095:Exoribonucleases; C022535:exoribonuclease II