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

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

Identification of Novel Serodiagnostic Signatures of Typhoid Fever Using a Salmonella Proteome Array.

Current diagnostic tests for typhoid fever, the disease caused by Salmonella Typhi, are poor. We aimed to identify serodiagnostic signatures of typhoid fever by assessing microarray signals to 4,445 S. Typhi antigens in sera from 41 participants challenged with oral S. Typhi. We found broad, heterogeneous antibody responses with increasing IgM/IgA signals at diagnosis. In down-selected 250-antigen arrays we validated responses in a second challenge cohort (n = 30), and selected diagnostic signatures using machine learning and multivariable modeling. In four models containing responses to antigens including flagellin, OmpA, HlyE, sipC, and LPS, multi-antigen signatures discriminated typhoid (n = 100) from other febrile bacteremia (n = 52) in Nepal. These models contained combinatorial IgM, IgA, and IgG responses to 5 antigens (ROC AUC, 0.67 and 0.71) or 3 antigens (0.87), although IgA responses to LPS also performed well (0.88). Using a novel systematic approach we have identified and validated optimal serological diagnostic signatures of typhoid fever

Structure of RF1 bound to the ribosome.

<p>(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). <i>E. coli</i> numbering is used for RF1 residues. Hydrogen bonds are indicated by the dotted lines.</p

Concentration dependence of the observed rate of RF1 binding.

<p>(A) Concentration dependence of the observed rate for phase 1 of RF1 binding. Plots were fit to a linear equation to determine the association (<i>k</i>₁) and dissociation (<i>k</i>₋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).</p

Kinetics of peptide hydrolysis by RF1.

<p>(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.</p

Kinetics of wild type RF1 and RF1 mutants binding to the ribosome.

<p>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 (<i>k</i>_obs1 and <i>k</i>_obs2).</p

Thermodynamics and kinetics of RF1 mutants binding to the ribosome.

<p>*taken from 17.</p

Fluorescence assay for determining the K_D of RF1 binding to the ribosome.

<p>(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.</p