Skip to main content
. 2015 Apr 8;4:e04659. doi: 10.7554/eLife.04659

Figure 6. The NBD of Ssa proteins is essential for tRNA recognition.

(A) Label transfer assays with 32P-labeled tRNA-ProUGG were performed in the presence of various concentrations of RCMLA. The amount of RCMLA is shown as the molar ratio against Ssa proteins. (B) Label transfer assays with full-length Ssa-His6 fusions or GST-fusions with partial Ssa proteins. Left, radioimaging; right, CBB staining. Arrowheads indicate the bands of GST-fusions that received radioactivity. (C) Label transfer assays were carried out with wild-type or mutant forms of GST-Ssa1p-NBD (Ssa1p-NBD) or GST-Ssa2p-NBD proteins (Ssa2p-NBD). Quantitated data and raw gel images of a typical experiment are shown in the upper graph and the lower panels, respectively. The relative label transfer efficiency represents a ratio of label transfer of a mutant GST-Ssa-NBD to that of the corresponding wild type. The efficiency of the wild-type protein is set to 100%. All the experiments are done in triplicates, and error bars represent SDVs. Original gel images and individual quantification data for the label transfer assays in this figure will be found in Figure 6—source data 1.

DOI: http://dx.doi.org/10.7554/eLife.04659.017

Figure 6—source data 1. Zip file containing source data for Figure 6.
DOI: http://dx.doi.org/10.7554/eLife.04659.018
elife04659s005.zip (3.4MB, zip)
DOI: 10.7554/eLife.04659.018

Figure 6.

Figure 6—figure supplement 1. Mutations introduced into the NBD of Ssa proteins.

Figure 6—figure supplement 1.

(A) Sequence comparison among Ssa1p, Ssa2p, bovine Hsc70 (BtHsc70) and E. coli Hsp70 (DnaK). Asterisks, identical residues; colons, strongly conserved residues; periods, weakly conserved residues. Residues altered in mutants examined in Figure 6C by the label transfer assay are colored, and their positions in Ssa proteins are shown in boldface. (B) Mutation points are indicated in the structural model of the NBD of bovine Hsc70 (1–394 aa) (PDB ID, 2QWL; Jiang et al., 2007). The structure of bovine Hsc70 (1–394aa) is shown in a ribbon model. Side chains of mutated residues are shown in wire drawing with the same color-code in A. An ADP molecule bound to the nucleotide binding cleft was shown in the green surface model.
DOI: http://dx.doi.org/10.7554/eLife.04659.019