Figure 1.
Structure of RF1 bound to the ribosome.
(A) RF1 (green) bound to the ribosome (grey) in the ribosomal A site with P site tRNA (purple), E site tRNA (orange), and mRNA (pink). (B) Detailed view on the decoding site showing RF1 residues (green), base G530 of 16S rRNA (grey) and the stop codon UAA (pink). The structure figures were prepared from PDB file 3D5A using PyMol. (C) and (D) Close up of the interactions between the stop codon (pink) and the RF1 residues (green). E. coli numbering is used for RF1 residues. Hydrogen bonds are indicated by the dotted lines.
Figure 2.
Fluorescence assay for determining the KD of RF1 binding to the ribosome.
(A) Changes in relative fluorescence intensity after adding increasing concentrations of wild type RF1 (open diamonds) and RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) to ribosomes programmed with a UAA stop codon. (B) Changes in relative fluorescence intensity after adding increasing concentrations of wild type RF1 (open diamonds), and RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) to ribosomes programmed with a UGA stop codon. Representative titration experiments without standard deviations are shown and the data were fit to the quadratic equation (black line). The total RF1 concentrations added are indicated on the x-axis.
Table 1.
Thermodynamics and kinetics of RF1 mutants binding to the ribosome.
Figure 3.
Kinetics of wild type RF1 and RF1 mutants binding to the ribosome.
Representative stopped-flow time course of 1 μM wild type RF1 (A) and 1 μM RF1 mutants Q185A (B), R186A (C), T190A (D), and T198A (E) binding to ribosome. The time courses (grey trace) were transformed and fit to a double-exponential equation (black line) to determine the observed rates of RF1 binding (kobs1 and kobs2).
Figure 4.
Concentration dependence of the observed rate of RF1 binding.
(A) Concentration dependence of the observed rate for phase 1 of RF1 binding. Plots were fit to a linear equation to determine the association (k1) and dissociation (k−1) rate constants. (B) Concentration dependence of the observed rate for phase 2 of RF1 binding. Plots were fit to a linear equation. The standard errors from three independent experiments are shown. Indicated are wild type RF1 (open diamonds), RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares).
Figure 5.
Kinetics of peptide hydrolysis by RF1.
(A) Representative TLC displaying the time course of RF1-catalyzed release of [35S]-fMet from ribosome release complex. Labels indicate wild type RF1 and RF1 mutants. The final extent of peptide release by wild type and RF1 mutants were similar and separate filter binding studies showed that the extent of peptide release by wild type RF1 with UAA codon was >90%. (B) Graph showing the peptide release time course at saturating concentrations of wild type RF1 (open diamonds), RF1 mutants Q185A (filled circles), R186A (filled triangles), T190A (open circles), and T198A (open squares) are shown. Data were individually normalized and fit to a single-exponential equation (black line) to determine the rate of peptide release. Standard errors from at least three independent experiments are shown.