Figure 1.
Ribosome association of ProQ under native and dissociating conditions.
Polysome profiles (254 nm) from wild type cell extracts in (A) associating conditions (10 mM Mg2+) and (B) dissociating conditions (1 mM Mg2+) are shown. Western-blot analysis of TCA-precipitated fractions using antibodies to ProQ, small ribosomal subunit protein 2 (S2), and large ribosomal subunit protein 3 (L3) are shown and aligned to the UV-absorbance trace. Whole cell extracts from wild type (BW25113) and ΔproQ (JM6733) are included, as is the soluble lysate (Load). UV-absorbance peaks correspond to (L to R): Free RNA/protein, 30S, 50S, 70S, and polysomes.
Figure 2.
Ribosome association of plasmid encoded proQ mutant constructs in polysome profiles.
(A) Schematic representation of proQ mutant constructs expressed from the IPTG inducible plasmid pCA24N. The predicted RNA binding region is colored in black, and amino-acid boundaries of each construct are labeled. (B) Mutant constructs were expressed as the lone copy of proQ. Western-blot analysis of TCA-precipitated fractions from the separation of ribosomal moieties on sucrose density gradients was performed. Whole cell extracts of cells after induction were included (Induced) along with soluble lysate (Load). Position of the 30S, 50S, 70S, and Polysome species are indicated based on western-blot localization of S2 (data not shown).
Figure 3.
Ribosome association of ProQ after mRNA disruption.
(A) Cell lysates were left untreated (solid line) or treated with limited MNase (dashed line) and ribosomal species separated on sucrose gradients. The positions of ProQ and ribosomal protein S2 in the resulting TCA-precipitated fractions is determined by western-blot analysis. (B) mRNA free 70S ribosomes were mixed with equimolar amounts of purified ProQ without (foreground) or with (background) the addition of equimolar P2 mRNA before application to sucrose gradients. The position of ProQ and ribosomal protein S2 in the resulting TCA-precipitated fraction is determined by western-blot analysis.
Figure 4.
mRNA binding kinetics monitored by native tryptophan fluorescence.
(A) 20 nM ProQ was titrated with increasing amounts of in vitro transcribed mRNAs (“●” proP P1, “▲” proP P2, “■” rpoS). Tryptophan was excited at 295 nm and the fluorescence was monitored at 355 nm. The y-axis is provided in terms of normalized fluorescence (FLF/FLI), determined by dividing the fluorescence at each mRNA concentration (FLF) by the fluorescence for 20nM protein only (FLI). DNA yielded no measurable fluorescence shift and is included as a negative control (“□” DNA) (B) Summary of binding affinities of ProQ for in vitro transcribed mRNAs.
Figure 5.
Ribosome association of ProQ in various strain backgrounds.
Cell lysates from various mutant backgrounds, as indicated, were separated by sucrose density ultracentrifugation and the localization of the ribosomal species (30S, 50S, 70s, polysomes) indicated. The localization of ProQ in the TCA-precipitated fractions is shown by immunoblot. Whole cell extracts from wild type, ΔproQ (JM6733), and the soluble lysate (Load) are included as indicated.
Figure 6.
Biofilm formation defect in a ΔproQ strain.
Wild type (BW25113), ΔproQ (JM6733), and ΔproP (JM6753) strains were examined for formation of biofilms after 6 days. Strains were transformed with either empty vector (pMR20; shaded) or vector containing the proQ open reading frame, plus 500 bp of genomic sequence upstream of the translation start site (pMR20-ProQ; unshaded). Error bars represent 95% confidence intervals. A statistical difference is indicated (*) between wild type and ΔproQ (JM6733) with a p-value < 0.001.