This entry represents the catalytic domain of ribonuclease II [ (PUBMED:16806266) ]. It includes characterised and related sequences to exoribonuclease II (RNase II) and ribonuclease R, a bacterial 3' --> 5' exoribonuclease homologous to RNase II [ (PUBMED:11948193) (PUBMED:15604703) (PUBMED:9829834) ].
Characterization of the functional domains of Escherichia coli RNase II.
J Mol Biol. 2006; 360: 921-33
Display abstract
RNase II is a single-stranded-specific 3'-exoribonuclease that degradesRNA generating 5'-mononucleotides. This enzyme is the prototype of anubiquitous family of enzymes that are crucial in RNA metabolism and sharea similar domain organization. By sequence prediction, three differentdomains have been assigned to the Escherichia coli RNase II: twoRNA-binding domains at each end of the protein (CSD and S1), and a centralRNB catalytic domain. In this work we have performed a functionalcharacterization of these domains in order to address their role in theactivity of RNase II. We have constructed a large set of RNase IItruncated proteins and compared them to the wild-type regarding theirexoribonucleolytic activity and RNA-binding ability. The dissociationconstants were determined using different single- or double-strandedsubstrates. The results obtained revealed that S1 is the most importantdomain in the establishment of stable RNA-protein complexes, and itselimination results in a drastic reduction on RNA-binding ability. Inaddition, we also demonstrate that the N-terminal CSD plays a veryspecific role in RNase II, preventing a tight binding of the enzyme tosingle-stranded poly(A) chains. Moreover, the biochemical results obtainedwith RNB mutant that lacks both putative RNA-binding domains, revealed thepresence of an additional region involved in RNA binding. Such region, wasidentified by sequence analysis and secondary structure prediction as athird putative RNA-binding domain located at the N-terminal part of RNBcatalytic domain.
