RNA-binding Site of Escherichia coli Peptidyl-tRNA Hydrolase

• Published in: The Journal of biological chemistry Vol. 286; no. 45; pp. 39585 - 39594
• Main Authors: Giorgi, Laurent, Bontems, François, Fromant, Michel, Aubard, Caroline, Blanquet, Sylvain, Plateau, Pierre
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 11.11.2011; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Catalysis; Chemical Sciences; Enzyme Mechanisms; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; NMR; Nuclear Magnetic Resonance, Biomolecular; Organic chemistry; Peptide Mapping; Protein Structure, Secondary; Protein Synthesis; Protein Synthesis and Degradation; 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; RNA-Protein Interaction; Transfer RNA (tRNA); Enzyme Mechanisms; NMR; Transfer RNA (tRNA); Protein Synthesis; RNA-Protein Interaction; Escherichia coli
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.281840

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 tRNAHis 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 tRNAHis. Catalytic efficiency of the hydrolase toward these two substrates is reduced by factors of 2 and 6, respectively, if compared with N-acetyl-histidyl-tRNAHis. 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. Background: Bacterial peptidyl-tRNA hydrolase is essential in recycling of ribosome-dissociated peptidyl-tRNAs. Results: Comparing minimalist substrates and using NMR mapping, the RNA-binding site of the hydrolase is characterized. Conclusion: Interaction between the hydrolase and tRNA involves features common to all elongator tRNAs. Significance: Knowledge of a bacterial peptidyl-tRNA hydrolase·substrate complex may drive the search for enzyme inhibitors.