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Thermodynamic and kinetic insights into stop codon recognition by release factor 1.

Trappl K, Mathew MA, Joseph S - PLoS ONE (2014)

Bottom Line: Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release.Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome.However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.

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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).
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pone-0094058-g004: 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).

Mentions: To determine the rate of stop codon recognition by RF1 with a cognate stop codon, transient-state kinetic studies of the various RF1 mutants were performed and compared to wild type RF1 [13], [17]. Time courses of RF1 binding to RC were determined using a stopped-flow instrument. Our previous study showed that the kinetics of RF1 binding has an initial fast phase followed by a second slower phase [17]. The amplitudes for the fast and slow phases are ≈20% and ≈10%, respectively. This biphasic increase in fluorescence could also be observed for the four RF1 mutants (Figure 3). The observed rates (kobs) of RF1 mutants binding to the ribosome were obtained by fitting the stopped-flow time courses to a double exponential equation. The experiment was performed at increasing concentrations of each RF1 mutants and a plot of RF1 concentration versus kobs for phase 1 was used to calculate the association rate constant (k1) and the dissociation rate constant (k−1) (Figure 4A). The association rate constants of the RF1 mutants were overall 2- to 14-fold reduced compared to wild type RF1 (68 μM−1 s−1 for wild type RF1). The association rate constant for RF1 mutants Gln185Ala, Arg186Ala, Thr190Ala and Thr198Ala are 15 μM−1 s−1, 5 μM−1 s−1, 33 μM−1 s−1 and 17 μM−1 s−1, respectively (Table 1). The RF1 His197Ala mutant studied previously has an association rate constant that is nearly identical to RF1 wild type (71 μM−1 s−1) [17]. The dissociation rate constant (k−1) was determined from the y-intercept of the concentration dependence plot of phase 1. The k−1 values are 22 s−1, 3 s−1, 12 s−1 and 48 s−1 for the RF1 mutants Gln185Ala, Arg186Ala, Thr190Ala and Thr198Ala, respectively. The k−1 value for the wild type RF1 is very small and due to the magnified error of extrapolation of the data appears to be negative in the plot. The k−1 values for the RF1 mutants are slower than the previously tested RF1 His197Ala mutant (k−1 = 175 s−1) [17]. Thus, the association rate constants of the RF1 mutants tested in this study are affected to a greater extent than the His197Ala mutant, whereas the dissociation rate constants appear to be less affected. The second phase observed in the stopped flow time course was previously assumed to be a first order conformational change after initial binding [17]. However, the plot of RF1 concentration versus kobs for phase 2 also showed concentration dependence and the reason for this unclear at the present time (Figure 4B). We could not determine the kinetics of RF1 mutants binding to RC with the non-cognate UGA codon because the apparent rate of association is very fast and most of the amplitude is lost in the deadtime of the instrument, which is consistent with the KD values in the micromolar range.


Thermodynamic and kinetic insights into stop codon recognition by release factor 1.

Trappl K, Mathew MA, Joseph S - PLoS ONE (2014)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3974865&req=5

pone-0094058-g004: 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).
Mentions: To determine the rate of stop codon recognition by RF1 with a cognate stop codon, transient-state kinetic studies of the various RF1 mutants were performed and compared to wild type RF1 [13], [17]. Time courses of RF1 binding to RC were determined using a stopped-flow instrument. Our previous study showed that the kinetics of RF1 binding has an initial fast phase followed by a second slower phase [17]. The amplitudes for the fast and slow phases are ≈20% and ≈10%, respectively. This biphasic increase in fluorescence could also be observed for the four RF1 mutants (Figure 3). The observed rates (kobs) of RF1 mutants binding to the ribosome were obtained by fitting the stopped-flow time courses to a double exponential equation. The experiment was performed at increasing concentrations of each RF1 mutants and a plot of RF1 concentration versus kobs for phase 1 was used to calculate the association rate constant (k1) and the dissociation rate constant (k−1) (Figure 4A). The association rate constants of the RF1 mutants were overall 2- to 14-fold reduced compared to wild type RF1 (68 μM−1 s−1 for wild type RF1). The association rate constant for RF1 mutants Gln185Ala, Arg186Ala, Thr190Ala and Thr198Ala are 15 μM−1 s−1, 5 μM−1 s−1, 33 μM−1 s−1 and 17 μM−1 s−1, respectively (Table 1). The RF1 His197Ala mutant studied previously has an association rate constant that is nearly identical to RF1 wild type (71 μM−1 s−1) [17]. The dissociation rate constant (k−1) was determined from the y-intercept of the concentration dependence plot of phase 1. The k−1 values are 22 s−1, 3 s−1, 12 s−1 and 48 s−1 for the RF1 mutants Gln185Ala, Arg186Ala, Thr190Ala and Thr198Ala, respectively. The k−1 value for the wild type RF1 is very small and due to the magnified error of extrapolation of the data appears to be negative in the plot. The k−1 values for the RF1 mutants are slower than the previously tested RF1 His197Ala mutant (k−1 = 175 s−1) [17]. Thus, the association rate constants of the RF1 mutants tested in this study are affected to a greater extent than the His197Ala mutant, whereas the dissociation rate constants appear to be less affected. The second phase observed in the stopped flow time course was previously assumed to be a first order conformational change after initial binding [17]. However, the plot of RF1 concentration versus kobs for phase 2 also showed concentration dependence and the reason for this unclear at the present time (Figure 4B). We could not determine the kinetics of RF1 mutants binding to RC with the non-cognate UGA codon because the apparent rate of association is very fast and most of the amplitude is lost in the deadtime of the instrument, which is consistent with the KD values in the micromolar range.

Bottom Line: Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release.Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome.However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California, United States of America.

ABSTRACT
Stop codon recognition is a crucial event during translation termination and is performed by class I release factors (RF1 and RF2 in bacterial cells). Recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain II of RF1/RF2. Additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of RF1 on the ribosome, which is essential for triggering peptide release. In this study we mutated four conserved residues in Escherichia coli RF1 (Gln185, Arg186, Thr190, and Thr198) that are proposed to be critical for discriminating stop codons from sense codons. Our thermodynamic and kinetic analysis of these RF1 mutants showed that the mutations inhibited the binding of RF1 to the ribosome. However, the mutations in RF1 did not affect the rate of peptide release, showing that imperfect recognition of the stop codon does not affect the proper positioning of RF1 on the ribosome.

Show MeSH
Related in: MedlinePlus