DnaK-facilitated ribosome assembly in Escherichia coli revisited

Abstract

Assembly helpers exist for the formation of ribosomal subunits. Such a function has been suggested for the DnaK system of chaperones (DnaK, DnaJ, GrpE). Here we show that 50S and 30S ribosomal subunits from an Escherichia coli dnaK-null mutant (containing a disrupted dnaK gene) grown at 30 degrees C are physically and functionally identical to wild-type ribosomes. Furthermore, ribosomal components derived from mutant 30S and 50S subunits are fully competent for in vitro reconstitution of active ribosomal subunits. On the other hand, the DnaK chaperone system cannot circumvent the necessary heat-dependent activation step for the in vitro reconstitution of fully active 30S ribosomal subunits. It is therefore questionable whether the requirement for DnaK observed during in vivo ribosome assembly above 37 degrees C implicates a direct or indirect role for DnaK in this process.

FIGURE 1.

(A) Kinetics of in vitro reconstitution of 30S ribosomal subunits at the indicated temperatures in the presence (closed symbols) or absence (open symbols) of the chaperones (DnaK, DnaJ, GrpE + ATP). At the indicated times, an aliquot was withdrawn from the reconstitution mixture, complemented with native 50S subunits and assayed for poly(Phe) synthesis at 20°C for 2 h. For details, see Materials and Methods. (B) ATPase activity of the DnaK system. The specific ATPase activity of DnaK (released phosphate/minute/DnaK) at 37°C was determined from the slope of the curve assuming a molecular mass for DnaK of 70 kD.

FIGURE 2.

Immunoanalysis of DnaK in native 50S and 30S ribosomal subunits. (Lane 1) Control, DnaK (30 ng ≅ 0.5 pmole); (lane 2) 5.2 A₂₆₀ units ≅ 375 pmole of 30S subunits from strain CAN20-12E (dnaK⁺); (lane 3) 4.4 A₂₆₀ units ≅ 160 pmole of 50S subunits from strain CAN20-12E; (lane 4) 5.6 A₂₆₀ units of 30S subunits from strain BB11 (ΔdnaK); (lane 5) 5.6 A₂₆₀ units of 50S subunits from strain BB11 (ΔdnaK). The subunits were boiled in SDS sample buffer before subjecting them to an SDS-gel electrophoresis followed by the blotting procedure. The relative density of the bands was determined by scanning (Molecular Dynamics) and processing with the ImageQuant program. The pixel numbers for lanes 1, 2, and 3 were 63,840, 2434, and 21,577, yielding a molar ratio of DnaK per 30S and 50S subunits of 1:20,000 and 1:950, respectively. For details, see Materials and Methods.

FIGURE 3.

Two-dimensional polyacrylamide gel electrophoresis of 50S and 30S ribosomal proteins from the dnaK⁺ (MC41.14) and ΔdnaK (BB11) strains. TP50 and TP30 were prepared and subjected to two-dimensional polyacrylamide gel electrophoresis according to Geyl et al. (1981). (A,B) TP30 from 30S subunits of the MC41.14 dnaK⁺ strain (wt-TP30) and mutant BB11 (ΔdnaK) strain (mu-TP30), respectively. (C,D) TP50 from 50S subunits of the MC41.14 dnaK⁺ strain (wt-TP50) and mutant BB11 (ΔdnaK) strain (mu-TP50), respectively.

FIGURE 4.

Sedimentation profiles of heat-treated 70S ribosomes prepared from dnaK⁺ (MC41.14) and ΔdnaK (BB11) strains. (A,B) Ribosomes from wild-type and mutant strains, respectively, were incubated at various temperatures (0°C, 30°C, 40°C, or 50°C) and then subjected to sucrose-gradient centrifugations. The profiles were practically identical; here those after a 50°C incubation are shown. (C,D) Same as A and B, respectively, but incubation temperature was 60°C; (E,F) same as A and B, respectively, but incubation temperature was 70°C. For details, see Materials and Methods.