Functional guanine–arginine interaction between tRNAPro and prolyl-tRNA synthetase that couples binding and catalysis

Abstract

SummaryAminoacyl-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.

Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain

“…In the case of ProXp-ala, which is significantly smaller than ProRS, the initial encounter does not involve anticodon interactions and both encounter and accommodation must rely exclusively on acceptor stem interactions. The proposed R80 side chain interaction with the major groove of G72 resembles the ProRS R144-G72 interaction reported earlier ( 56 ). We propose that the R80–G72 interaction contributes to the stabilization of the initial substrate encounter complex, with the K50 residue playing a more minor role.…”

“…The G72 nucleotide of bacterial tRNA Pro is a critical recognition element for both ProRS and the ProXp-ala trans -editing domain. Based on kinetic data and a novel cross-linking approach, we previously identified a specific hydrogen bonding interaction between an Arg side chain in the active site of E. coli ProRS (R144 in the conserved motif 2 loop) and the major groove functional groups of G72 ( 56 ). Changes at R144 did not substantially alter the Michaelis constant for tRNA, but significantly affected the k cat parameter.…”

miRNases: Novel peptide-oligonucleotide bioconjugates that silence miR-21 in lymphosarcoma cells

“…With the example of onconase, it was shown that replacement of Glu91 by an arginine residue in an onconase binding centre that specifically binds with guanine nucleobase in d(AUGA) increased the guanine preference and afforded an onconase variant with the highest known k cat /K M value [86]. A direct interaction between an arginine residue Arg144 in the active site of E. coli prolyl-tRNA synthetase and the G72 residue in the acceptor stem of tRNA Pro was found to be a key contact providing the allosteric interaction between the anticodon domain and the aminoacylation active site [87]. It was shown that the structural basis for RNA recognition by ZRANB2 protein was the formation of a special network of hydrogen bonds between the GGUcontaining ssRNA-motif and Arg81 and Arg82 of ZRANB2 via bidentate interactions of both Gua with side chains of arginines [88].…”

Influence of Sequential Guanidinium Methylation on the Energetics of the Guanidinium···Guanine Dimer and Guanidinium···Guanine···Cytosine Trimer: Implications for the Control of Protein···DNA Interactions by Arginine Methyltransferases

“…Arg residues are intimately involved in the interactions between many nucleic acid binding proteins and nucleic acid. − Among the different stabilizing interactions observed between the Arg residues of nucleic acid binding proteins and nucleic acids are the formation of hydrogen bonds between the Arg ω-NH 2 + protons and a guanine nucleic acid base. ,− In this case, the Arg residue will be oriented within the major groove of the DNA helix, and the guanine···Arg interaction will be mostly stabilized through the formation of two key hydrogen bonds, those between the Arg ω-NH 2 groups and the guanine carbonyl oxygen and imine nitrogen groups (Figure ). Arg methylation will undoubtedly influence the energetics of this key intermolecular interaction.…”