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Role of N ‐terminal extension of B acillus stearothermophilus RN ase H 2 and C ‐terminal extension of T hermotoga maritima RN ase H 2
Author(s) -
Permanasari EtinDiah,
Angkawidjaja Clement,
Koga Yuichi,
Kanaya Shigenori
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12479
Subject(s) - thermotoga maritima , aquifex aeolicus , rnase p , substrate (aquarium) , stereochemistry , crystallography , chemistry , biology , biochemistry , rna , escherichia coli , gene , ecology
Bacillus stearothermophilus RN ase H 2 ( B st RNH 2) and Thermotoga maritima RN ase H 2 ( T ma RNH 2) have N ‐terminal and C ‐terminal extensions, respectively, as compared with Aquifex aeolicus RN ase H 2 ( A ae RNH 2). To analyze the role of these extensions, B st RNH 2 and T ma RNH 2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and A ae RNH 2. The far‐ UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RN ase H activities of B st RNH 2 and T ma RNH 2, as well as their substrate‐binding affinities, were considerably decreased by removal of these extensions. The stability of B st RNH 2 and T ma RNH 2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of T ma RNH 2 without the C‐terminal 46 residues were partly restored by the attachment of the N ‐terminal extension of B st RNH 2. These results suggest that the N ‐terminal extension of B st RNH 2 functions as a substrate‐binding domain and stabilizes the RN ase H domain. Because the C ‐terminal extension of T ma RNH 2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate‐binding site functional. A ae RNH 2 showed comparable junction ribonuclease activity to those of B st RNH 2 and T ma RNH 2, and was more stable than these proteins, indicating that bacterial RN ases H 2 do not always require an N ‐terminal or C ‐terminal extension to increase activity, substrate‐binding affinity, and/or stability.