title: RNA-binding site of Escherichia coli peptidyl-tRNA hydrolase.
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.
authors.affiliation: Ecole Polytechnique, Laboratoire de Biochimie, CNRS, 91128 Palaiseau Cedex, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Giorgi
authors.forename: François
authors.initials: F
authors.lastname: Bontems
authors.forename: Michel
authors.initials: M
authors.lastname: Fromant
authors.forename: Caroline
authors.initials: C
authors.lastname: Aubard
authors.forename: Sylvain
authors.initials: S
authors.lastname: Blanquet
authors.forename: Pierre
authors.initials: P
authors.lastname: Plateau
mesh_terms: D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012354:RNA, Transfer, His / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012354:RNA, Transfer, His; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase