PMID:21930710
| Citation |
Giorgi, L, Bontems, F, Fromant, M, Aubard, C, Blanquet, S and Plateau, P (2011) RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase. J. Biol. Chem. 286:39585-94 |
|---|---|
| Abstract |
In a cell, peptidyl-tRNA molecules that have prematurely dissociated from ribosomes need to be recycled. This work is achieved by an enzyme called peptidyl-tRNA hydrolase. To characterize the RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase, minimalist substrates inspired from tRNA(His) have been designed and produced. Two minisubstrates consist of an N-blocked histidylated RNA minihelix or a small RNA duplex mimicking the acceptor and TψC stem regions of tRNA(His). Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNA(His). In contrast, with an N-blocked histidylated microhelix or a tetraloop missing the TψC arm, efficiency of the hydrolase is reduced 20-fold. NMR mapping of complex formation between the hydrolase and the small RNA duplex indicates amino acid residues sensitive to RNA binding in the following: (i) the enzyme active site region; (ii) the helix-loop covering the active site; (iii) the region including Leu-95 and the bordering residues 111-117, supposed to form the boundary between the tRNA core and the peptidyl-CCA moiety-binding sites; (iv) the region including Lys-105 and Arg-133, two residues that are considered able to clamp the 5'-phosphate of tRNA, and (v) the positively charged C-terminal helix (residues 180-193). Functional value of these interactions is assessed taking into account the catalytic properties of various engineered protein variants, including one in which the C-terminal helix was simply subtracted. A strong role of Lys-182 in helix binding to the substrate is indicated. |
| Links |
PubMed PMC3234781 Online version:10.1074/jbc.M111.281840 |
| Keywords |
Binding Sites; Carboxylic Ester Hydrolases/chemistry; Carboxylic Ester Hydrolases/genetics; Carboxylic Ester Hydrolases/metabolism; Catalysis; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Nuclear Magnetic Resonance, Biomolecular; Peptide Mapping; Protein Structure, Secondary; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Transfer, Amino Acyl/chemistry; RNA, Transfer, Amino Acyl/genetics; RNA, Transfer, Amino Acyl/metabolism; RNA, Transfer, His/chemistry; RNA, Transfer, His/genetics; RNA, Transfer, His/metabolism |
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