Figure 1.
Purification and EMSA of TRBP-WT protein.
(A) Coomassie brilliant blue stained pattern of purified TRBP-WT protein resolved by SDS-PAGE. Arrowhead indicates 44 kDa of TRBP-WT protein. M represents the protein size markers. (B–D) The results of EMSA of TRBP-WT protein with 32P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). 32P-labeled siRNA (0.50 nM) was incubated with increasing amounts of TRBP-WT protein, as indicated. Lower cases in siLuc-36D sequence in D indicate DNAs. (E) Supershift analysis of TRBP-WT protein (1.3 nM) with no antibodies, control anti-Flag, and anti-myc antibodies. (F) The result of EMSA of TRBP-WT protein (1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM) incubated with increasing amount of non-labeled siLuc-36. In B–F, arrows indicate positions of the first and second step migrating complexes, corresponding to TRBP-WT complexes 1 and 2, respectively, and the supershifted complex, in addition to siRNA and ATP.
Table 1.
The Kd values (nM) of TRBP-WT and PACT-WT and their mutants.
Figure 2.
Gel filtration chromatography of purified TRBP-WT and TRBP-ΔdsRBD3 proteins with siRNA.
Gel filtration chromatography patterns of non-labeled 300 nM of siLuc-36 alone (A), siLuc-36 with 1,300 nM of TRBP-WT (B), siLuc-36 with 5.1 nM of TRBP-WT(C) and siLuc-36 with 1,300 nM of TRBP-ΔdsRBD3 (D). In A, inset, siLuc-36 was electrophoresed on a 3% agarose gel and stained with EtBr. Lower panels in B-D showed the results of Western blot (WB) by anti-myc antibody for detecting TRBP proteins in the elution fractions. Histograms below WBs showed the quantified signal densities of TRBP proteins detected by WB. Arrowheads indicate the positions of molecular weight size markers. In B, each fraction of molecular weight size markers was quantified and represented in histogram after staining the gel with Coomassie brilliant blue.
Figure 3.
Structure, purification and EMSA of TRBP mutant proteins.
(A) Schematic representation of TRBP-WT and its mutant proteins. Amino acids at positions 80 and 81 were substituted from lysines (KK) to alanines (AA) in TRBP-dsRBDmt1, and those at 210 and 211 were also substituted from KK to AA in TRBP-dsRBDmt2. TBRP-dsRBDmt1+2 contains both substitutions. The dsRBD3 was deleted in TRBP-ΔdsRBD3. All mutants were tagged with N-terminal His and C-terminal myc, in the same way as TRBP-WT. (B) Coomassie brilliant blue stained patterns of purified TRBP mutant proteins resolved by SDS-PAGE. Arrows indicate the migration position of TRBP-dsRBDmt1, TRBP-dsRBDmt2, and TRBP-dsRBDmt1+2. Arrowhead indicates TRBP-ΔdsRBD3 protein. M represents the protein size markers. (C–F) EMSA patterns of TRBP-dsRBDmt1 (C), TRBP-dsRBDmt2 (D), TRBP-dsRBDmt1+2 (E) and TRBP-ΔdsRBD3 (F) with 32P-labeled siLuc-36. 32P-labeled siRNA (0.50 nM) was incubated with increasing amounts of TRBP protein, as indicated. (G) The comparison of mobilities of siRNA complexes with TRBP-WT, TRBP-dsRBDmt1, TRBP-dsRBDmt2, TRBP-dsRBDmt1+2 and TRBP-ΔdsRBD3 proteins (1,300 nM), respectively. (H) EMSA pattern of TRBP-ΔdsRBD3 protein (1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM) incubated with increasing amount of non-labeled siLuc-36.
Figure 4.
Purification of PACT-WT and its mutant proteins, and EMSA of PACT-WT protein.
(A) Coomassie brilliant blue stained pattern of purified PACT-WT (left panel), and its mutant proteins (right panel) resolved by SDS-PAGE. Arrows indicate the migration position of PACT-WT, PACT-dsRBDmt1, PACT-dsRBDmt2, and PACT-dsRBDmt1+2 proteins. Arrowhead indicates PACT-ΔdsRBD3 protein. M represents the protein size markers. (B–D) EMSA patterns of PACT-WT with 32P-labeled siLuc-36 (B), siLuc-36B (C), and siLuc-36D (D). 32P-labeled siRNA (0.50 nM) was incubated with increasing amounts of PACT-WT as indicated. Lower cases in D indicate DNAs.
Figure 5.
Structure and EMSA of PACT-WT and PACT mutant proteins.
(A) Schematic representation of PACT-WT and its mutant proteins. Amino acids at positions 84 and 85 were substituted from lysines (KK) to alanines (AA) in PACT-dsRBDmt1, and those at 177 and 178 were also substituted from KK to AA in PACT-dsRBDmt2. PACT-dsRBDmt1+2 contained both substitutions. The dsRBD3 was deleted in PACT-ΔdsRBD3. All mutants were tagged with N-terminal His and C-terminal myc, in the same way as PACT-WT. (B–E) EMSA patterns of PACT-dsRBDmt1 (B), PACT-dsRBDmt2 (C), PACT-dsRBDmt1+2 (D), and PACT-ΔdsRBD3 (E) with 32P-labeled siLuc-36. (F, G) EMSA patterns of PACT-WT (F) and PACT-ΔdsRBD3 (G) (1,300 nM) mixed with 32P-labeled siLuc-36 (0.50 nM) incubated with increasing amount of non-labeled siLuc-36.
Figure 6.
Gel filtration chromatography of purified PACT-WT and PACT mutant proteins with siRNA.
Gel filtration chromatography patterns of non-labeled siLuc-36 (300 nM) with PACT-WT (A), PACT- dsRBDmt1 (B), PACT-dsRBDmt2 (C), and PACT-ΔdsRBD3 (D). Lower panels showed the results of Western blot (WB) by anti-myc antibody for detecting PACT proteins in the elution fractions. Histograms below WBs showed the quantified signal densities of PACT proteins detected by WBs. Arrowheads indicate the positions of molecular weight size markers.
Figure 7.
Model of TRBP and PACT binding to siRNA.
(A, B) TRBP-WT (A) or TRBP-ΔdsRBD3 (B) binds one molecule of siRNA as a monomer at low concentrations, and then each protein dimerizes due to the increased protein concentration. However, excessive amount of siRNAs were added, TRBP-WT or TRBP-ΔdsRBD3 dimer was dissociated into monomeric TRBP-WT or TRBP-ΔdsRBD3 dimer containing a single molecule of siRNA. (C) PACT-WT forms homodimers at high concentrations and binds to one or two molecules of siRNA. (D) PACT-ΔdsRBD3 binds one siRNA molecule as a monomer or binds one or two siRNA molecules as a dimer. The monomer and dimer may achieve equilibrium, although the monomeric form is predominant. In C and D, we could not determine whether the siRNA shown in gray is contained in the dimerized PACT proteins or not.