(A) Screening of KH mutant Der proteins and their phenotypes in ΔrrmJ. The C-terminal part of the der gene was randomly mutagenized by PCR as described by Lerner et al. (18) and Spee et al. (25). The four deoxynucleoside triphosphates (dNTPs) (dATP, dCTP, dGTP, and dTTP) were depleted one at a time in PCR. MnCl₂ (0.5 mM) and dITP (200 μM) were also included in the reaction, and error-prone Taq polymerase was used. Plasmids with mutations were transformed into ΔrrmJ, and transformants were streaked onto LB plates containing ampicillin with or without 1 mM IPTG. (B) Growth curves of ΔrrmJ transformed with KH mutant plasmids. Cell culture and optical density measurement were carried out as described for Fig. 1B.

Phenotypes of der-deletion strain expressing KH mutant Der. (A) The der-deletion strain was transformed with pINEcDer, pINEcDerV400A, pINEcDerG414R, pINEcDerG424D, pINEcDerN469K, pINEcDerT472A, or pINEcDer460 and plated on LB plates containing chloramphenicol (40 μg/ml) and kanamycin (35 μg/ml). Plates were first incubated at 30°C, and colonies formed at 30°C were streaked onto LB plates containing chloramphenicol and kanamycin; plates were then incubated at 42°C. (B) The nonspecific interaction of Der460 with ribosomes. Wild-type cells harboring pIN^AEcDer or pIN^AEcDer460 were cultured, and polysome samples were prepared in the presence of GDPNP, GDP, or Apo state. Polysome and Western blot analyses were carried out as described for Fig. 1. The wild-type cells harboring pIN^AEcDer or pIN^AEcDer460 were used as controls (shown in the first two lanes of the Western blot). An arrowhead indicates the endogenous Der in 50S subunit fractions, and the numbers indicate the ends of each fraction.

Phenotypes of ΔrrmJ expressing G domain and mutant Der. (A) The ΔrrmJ (strain HB23) cells transformed with plasmids were grown at 30°C overnight in LB medium containing ampicillin (50 μg/ml), and cultures were diluted. The diluted cultures were streaked on LB plates containing ampicillin with or without 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside). GD-1, GD-2, GA-1, and GA-2 indicate N118D, N321D, S16A, and S216A mutations, respectively. GD-12 and GA-12 indicate the double mutations. (B) Growth curves of ΔrrmJ harboring different plasmids. Cells were cultured in LB medium containing ampicillin at 30°C, and cell cultures were diluted five times before measurement of the optical density at 600 nm (OD₆₀₀). (C) Polysome profiles of wild-type and G-domain mutant Der in ΔrrmJ. The ΔrrmJ cells harboring various plasmids were cultured to the log phase at 30°C in LB medium containing ampicillin. Polysomes were trapped by the addition of chloramphenicol to the culture to achieve a final concentration of 0.1 mg/ml. The cell pellets were resuspended with BP buffer (20 mM Tris-HCl [pH 7.5], 10 mM MgCl₂, 100 mM NH₄Cl, and 5 mM β-mercaptoethanol). GDPNP was added at a final concentration of 100 μM. Approximately 10 A₂₆₀ units of each cleared extract was layered onto a sucrose gradient. The preparation of cell lysates and polysome analysis were carried out by ultracentrifugation in a Beckman SW41 rotor for 3.5 h at 210,000 × g. as described previously (15). (D) Association of wild-type and G-domain mutant Der with 50S subunits in ΔrrmJ. Individual gradient fractions (16 μl) prepared as described for panel C were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis followed by Western blotting using anti-Der antisera (15).

Ribosome profiles of ΔrrmJ expressing KH mutant Der. (A) The ΔrrmJ cells harboring the indicated plasmids were cultured at 30°C in LB medium containing ampicillin, and polysomes were resolved by ultracentrifugation carried out as described for Fig. 1. (B) Cofractionation of mutant Der with 50S subunits in ΔrrmJ. SDS-PAGE analysis and Western blotting were carried out as described for Fig. 1D. Western blots of three controls were taken from the experiment represented in Fig. 1D. (C) Ribosomal subunit profiles of ΔrrmJ. The cell pellets were resuspended with BP buffer containing 0.25, 0.5, or 1 mM MgCl₂. Polysomes were resolved by ultracentrifugation for 3.5 h at 230,000 × g. Three lines (a, b, and c) indicate aberrant 50S subunits with three different migration patterns. Arrows indicate 50S subunits. (D) Ribosomal subunit patterns of ΔrrmJ expressing RrmJ, Der, and the Der KH mutant. The cell pellets were prepared as described for panel A and resuspended in BP buffer containing 0.25 mM MgCl₂. Subunits were resolved by ultracentrifugation. An arrowhead indicates the position of aberrant 50S subunits.