Fig. 1: mjCgi121 is the tRNA-binding subunit of arKEOPS.
From: A substrate binding model for the KEOPS tRNA modifying complex
a, b Binding activity of arKEOPS, individual subunits, and subcomplexes towards mjtRNALys assessed using a filter binding assay. Protein concentrations and the specific arKEOPS proteins tested are indicated. c Binding activity of arKEOPS mjCgi121-containing complexes at 1 µM concentration towards full length mjtRNALys, sctRNAIle, or their respective anticodon stem loops (ASL) assessed using the filter binding assay. d (Left) Nuclear magnetic resonance 1H-15N-HSQC analysis of 15N-labeled mjCgi121 in the presence or absence of 1:0.1 molar ratio of mjtRNALys. (Right) Chemical shift perturbations (CSPs) and the peak intensity changes of assigned resonances are shown. Residues with CSP ≥ 0.02 ppm (top graph) or peak intensity changes of ≤0.4 (bottom graph) were considered hyper-perturbed by the tRNA. Thresholds are highlighted on graphs by horizontal dashed green lines. Hyper-perturbed residues and their projections on a schematic representation of the secondary structure of mjCgi121 (bottom) are highlighted in red. e Projection of hyper-perturbed residues from (d) in red on the surface structure of mjCgi121 (PDB 3ENH) identifies a putative tRNA-binding surface. Bud32 (green ribbon) was shown previously to bind to a site remote from the identified tRNA-binding surface. For clarity, only mjCgi121 and part of mjBud32 are shown (residues 344-419 of the naturally occurring mj1130 Kae1-Bud32 fusion protein).
