Functional guanine-arginine interaction between tRNAPro and prolyl-tRNA synthetase that couples binding and catalysis.
Aminoacyl-tRNA synthetases catalyze the attachment of specific amino acids to their cognate tRNAs. Specific aminoacylation is dictated by a set of recognition elements that mark tRNA molecules as substrates for particular synthetases. Escherichia coli prolyl-tRNA synthetase (ProRS) has previously been shown to recognize specific bases of tRNA(Pro) in both the anticodon domain, which mediate initial complex formation, and in the acceptor stem, which is proximal to the site of catalysis. In this work, we unambiguously define the molecular interaction between E. coli ProRS and the acceptor stem of cognate tRNA(Pro). Oxidative cross-linking studies using 2'-deoxy-8-oxo-7,8-dihydroguanosine-containing proline tRNAs identify a direct interaction between a critical arginine residue (R144) in the active site of E. coli ProRS and the G72 residue in the acceptor stem of tRNA(Pro). Assays conducted with motif 2 loop variants and tRNA mutants wherein specific atomic groups of G72 were deleted, are consistent with a functionally important hydrogen-bonding network between R144 and the major groove of G72. These results taken together with previous studies suggest that breaking this key contact uncouples the allosteric interaction between the anticodon domain and the aminoacylation active site, providing new insights into the communication network that governs the synthetase-tRNA interaction.
Department of Chemistry, University of Minnesota, 207 Pleasant Street Southeast, Minneapolis, MN 55455, USA.
Brian
B
Burke
Songon
S
An
Karin
K
Musier-Forsyth
D019943:Amino Acid Substitution; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012361:RNA, Transfer, Pro / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012361:RNA, Transfer, Pro; D011994:Recombinant Proteins; D006147:Guanine; D001120:Arginine; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase