The carboxy-terminal extension of the zinc-finger domain in YjeQ is necessary for its function in vivo. (A) The parental strain and the yjeQ null strain by itself or complemented with the plasmid expressing yjeQ, YjeQ M3 variant, or the empty vector were induced at t = 0 h with the indicated concentration of IPTG and grown at 25°C for 47 h. Growth was monitored by measuring absorbance at 600 nm and plotted against time. Standard deviations shown in the plot correspond to three replicas of the experiment. (B) Total rRNA of cell cultures were analyzed when cultures reached mid-log phase represented by OD600 = 0.2. Total rRNA was extracted and resolved by electrophoresis in 0.9% synergel–0.7% agarose gel. The marker (M) is in base pairs. (C) Ribosome absorbance profiles from the parental strain and yjeQ null strain by itself or complemented with the plasmid expressing YjeQ, YjeQ M3 variant, or the empty plasmid were fractionated by ultracentrifugation in 10%–30% sucrose gradients providing the profiles shown in this panel. Peaks for the 30S, 50S subunits, and 70S ribosomes are indicated. The proportion of free 30S to bound 30S (i.e., 30S subunits in 70S complexes) in each case was calculated by integrating the areas under the 30S and 70S peaks of the sucrose gradient profiles. The area of the 30S peak plus one-third the area of the 70S peak corresponds to the total 30S population. The area of the 30S peak was divided by the total 30S absorbance to obtain the percentage of free 30S subunits and produce the pie charts in this panel. The standard deviations shown correspond to three replicas of the experiment. Peak area for the 30S subunit in each case was measured with respect to the area under the 70S peak to calculate the percentage of free 30S subunits in both strains. The calculated percentages are shown in the pie charts. The standard deviations shown correspond to three replicas of the experiment.
