Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system

Abstract

Recently, there has been controversy regarding the ability of the DnaK chaperone system to facilitate Escherichia coli 30S subunit assembly at otherwise nonpermissive conditions. Here, we present additional data indicating that purified DnaK chaperone assembled 30S subunits are functional. Additionally, explanations for reported differences are discussed.

FIGURE 1.

Western blot analysis of in vitro reconstituted 30S particles. Reconstituted 30S subunits were formed at 15°C under the following conditions: 16S rRNA:DnaK:DnaJ:GrpE 1:1:1:2. The resulting particles were applied to 10%–40% sucrose gradients (Culver and Noller 1999); the peak was collected and split into two equal fractions. One fraction was directly precipitated, whereas the second was concentrated and washed (with a buffer containing 330 mM KCl) on a Centricon 100 sieving filter prior to precipitation. Equal fractions were probed with monoclonal anti-DnaK antibody from StressGen Biotech. (Lane 1) 50 ng of purified DnaK from StressGen Biotech. (Lane 2) Sucrose-gradient purified reconstituted 30S particle. (Lane 3) Sucrose-gradient purified and Centricon 100 treated reconstituted 30S particle.

FIGURE 2.

Western blot analysis of Escherichia coli extracts for the DnaK chaperone system. Control, 0.5 μg of purified protein (DnaK, DnaJ, or GrpE) purchased from StressGen Biotech. S100, 20 μg S100 extract; S150, 20 μg S150 extract. (Lane 1) Membrane probed with monoclonal anti-DnaK antibody. (Lane 2) Membrane probed with polyclonal anti-DnaJ antibody. (Lane 3) Membrane probed with polyclonal anti-GrpE antibody. All antibodies were purchased from StressGen Biotech. S100 prepared as reported in (Maki et al. 2002). S150 prepared as reported by Nierhaus (1990).

FIGURE 3.

Polyphenylalanine synthesis by purified in vitro reconstituted Escherichia coli 30S particles. Reconstitutions were performed under the conditions of Alix and Nierhaus (2003). Polyphenylalanine generated by purified 30S particles reconstituted under normal, high-temperature (42°C) conditions (solid circles) compared to natural 50S subunits alone (open squares), or particles reconstituted at low temperatures (20°, 21S) (solid diamonds), or as for the 21S reconstitution but in the presence of the DnaK chaperone system (solid squares). Chaperone conditions were similar to those of Maki et al. (2002) (16S rRNA:DnaK:DnaJ:GrpE 1:1:1:2 with 1mM ATP). The polyphenylalanine synthesis was carried out essentially as described (Southworth et al. 2002), by incubating 30S particles and 50S subunits (0.3 μM) with poly-uridine (0.35 mg/mL), ¹⁴C Phe-tRNA^Phe, His-tagged EF-G (0.3 μM), His-tagged EF-Tu (2 μM), GTP (1.4 mM), phosphoenolpyruvate (3.5 mM), and pyruvate kinase (14 μg/mL). Reactions were carried out in 80 mM K⁺-HEPES pH 7.6, 13 mM MgCl₂, and 100 mM KCl.