GTPase activity of <i>P. aeruginosa</i> EF-G1A and EF-G1B.

<p>Shown are representative GTPase assays of EF-G and ribosome dependence and a graph showing the linear increase in GTPase activity between 1 and 30 min. Assays are as described under “Methods and Materials”. A) Activity of EF-G1A and EF-G1B (1.0 and 0.3 μM, respectively) in the presence of varying concentrations of <i>P. aeruginosa</i> ribosomes. B) GTPase activity of EF-G at varied concentrations in the presence of 0.4 μM ribosomes. C) GTPase activity at increasing times. EF-G1A and EF-G1B are represented by filled diamonds (♦) and squares (■), respectively.</p

RRF and EF-G1A function to reduce the synthesis of poly(Phe).

<p>Representative assays depicting the effect of ribosome release factor (RRF) on the activities of EF-G1A, EF-G1B and a combination of EF-G1A and EF-G1B. A) Assays to determine the effect of RRF on the activity of EF-G1B were as described under “Methods and Materials”, with RRF titrated into the assay between 0.2 and 6.4 μM. The concentration of EF-G1B was 0.2 μM. B) Assays to determine the effect of RRF/EF-G1A on the activity of EF-G1B. The concentration of EF-G1B was the same as in “A” and the concentration of EF-G1A and RRF were 1 and 2 μM, respectively.</p

The effect of fusidic acid on the GTPase activity of <i>P. aeruginosa</i> EF-G1A and EF-G1B.

<p>Representative GTPase assays of the activity of EF-G1A and EF-G1B in increasing amounts of fusidic acid. The concentration of EF-G1A and EF-G1B were 1.0 and 0.3 μM, respectively, and the concentration of ribosomes was 0.4 μM. FA was added to the assay in concentrations from 4 μM to 250 μM. EF-G1A and EF-G1B are represented by filled diamonds (♦) and squares (■), respectively.</p

Determination of kinetic parameters for the GTPase activity of <i>P. aeruginosa</i> EF-G1B.

<p>A: Initial velocities for <i>P. aeruginosa</i> EF-G1B in GTPase activity reactions were determined at various concentrations of GTP. The concentration of EF-G1B was held constant at 0.3 μM. The velocities were measured between 1 and 6 min to minimize the chance of measurement of GTP hydrolysis occurring during mixing but before the beginning of the incubation period. The reactions were at 37 °C. The concentrations of GTP were: ♦, 25 μM; ■, 50 μM; ▲, 100 μM; +, 200 μM; ●, 400 μM, ×, 600 μM. B: The data from the initial velocity experiments were used to develop a Lineweaver-Burk plot to determine kinetic parameters for the GTPase activity of <i>P. aeruginosa</i> EF-G1B.</p

The effect of fusidic acid on the activity of <i>P. aeruginosa</i> EF-G in protein synthesis.

<p>Representative assays of the effect of increasing concentrations of fusidic acid on the ability of EF-G1A and EF-G1B to function in protein synthesis. A) EF-G1A and B) EF-G1B. The concentration of EF-G1A and EF-G1B were 1.0 and 0.2 μM, respectively, and the concentration of ribosomes was 0.2 μM. FA was added to the assay in concentrations from 4 μM to 500 μM. EF-G1A and EF-G1B are represented by filled diamonds (♦) and squares (■), respectively.</p

An alignment of EF-G1A and EF-G1B from <i>P. aeruginosa</i>.

<p>The protein sequences were downloaded from the National Center for Biotechnology Information (NCBI). The accession numbers for the two sequences are AAG05459 for EF-G1B and AAG07654 for EF-G1A. Sequence alignments were performed using Vector NTI Advance (TM) 11.0 (Invitrogen). Domains are designated with solid arrows and the G’ insert is designated with dotted arrows. Resistance mutations induced by fusidic acid at conserved amino acid residues are shown as closed circles (●) and mutations at invariant residues are shown as open circles (o). The amino acid representing the Walker B box is shown as (▼).</p

The ability of both forms of <i>P. aeruginosa</i> EF-G to function in protein synthesis.

<p>Representative protein synthesis assays containing increasing concentrations of EF-G1A and EF-G1B. The assays were as described under “Methods and Materials”. Concentrations of EF-G were as shown and concentrations of ribosomes were held constant at 0.2 μM. EF-G1A and EF-G1B are represented by filled diamonds (♦) and squares (■), respectively. “Phe Incorporated” represents the amount of phenylalanine incorporated into peptides during protein synthesis. Background activity (0.5 μM) was subtracted from the assay containing EF-G.</p

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified <i>P. aeruginosa</i> EF-G1A and EF-G1B.

<p>Samples (1.0 μg) of the <i>P. aeruginosa</i> EF-G1A and EF-G1B preparations were analyzed on a 4-20% SDS-PAGE gel and the protein bands were visualized by staining with Coomassie blue.</p