Cloning, expression and purification of HflX.
Recombinant E. coli
HflX and deletion constructs ΔN-HflX, ΔC-HflX and HflX-G proteins were cloned, expressed and purified to homogeneity (for details, see supplementary material
). Purified proteins were concentrated, aliquoted and stored at −80 °C after snap freezing in liquid nitrogen, for further use.
Gel retardation assays. These assays were performed by incubating HflX with 16S and 23S rRNA at 37 °C for 15 min in the presence or absence of GMPPNP/GDP (Sigma–Aldrich). Twenty percent Glycerol was added to the reaction mixture and was analyzed by native agarose gel stained with ethidium bromide.
Ribosome purification. BL21 cells, grown till 0.6 OD600 at 37 °C and incubated on ice for 10 min after addition of 100 μg/mL chloramphenicol, were harvested by centrifugation and were lysed by 5 cycles of freeze-thaw in Buffer D (20 mM Tris–HCl pH 8.0, 50 mM NH4Cl, 5 mM MgCl2, 1 mM DTT, 1 mg/mL lysozyme, protease inhibitor cocktail). Following the addition of RNase free DNase, the lysate was clarified by centrifugation at 45,000g at 4 °C. Supernatant was loaded on a 1.1 M sucrose cushion and centrifuged at 50,000 RPM for 4 h at 4 °C (Sorvall-TH660 rotor). The pellet was dissolved in Buffer E (20 mM Tris–HCl pH 8.0, 50 mM NH4Cl, 1 mM Mg-Acetate, 1 mM DTT). The ribosome thus obtained was stored at −80 °C.
Ribosomal subunits were purified by loading the supernatant (as mentioned above) on 18–50% sucrose step gradient and centrifuged at 28,000 RPM (Sorvall Surespin-630 rotor) at 4 °C for 10 h. Gradient was fractionated by upward displacement using 60% sucrose by ISCO density gradient fractionator. RNA isolated from each fraction was analyzed by formaldehyde agarose gel (1.5% agarose). Fractions containing 30S and 50S subunits were identified based on the presence of 16S and 23S rRNA, respectively. Fractions corresponding to these subunits were pooled separately and diluted by the addition of Buffer F (20 mM Tris–HCl pH 8.0, 50 mM NH4Cl, 10 mM Magnesium Chloride, 1 mM DTT). Ribosomal subunits were then concentrated using Millipore Amicon ultra centrifugal filter tubes.
While using them in ATP/GTP hydrolysis assays, 50S was diluted in Buffer G (20 mM Tris–HCl pH 8.0, 200 mM NH4Cl, 1 mM Mg-Acetate, 1 mM DTT) and precipitated by centrifuging at 45,000 RPM at 4 °C for 4 h (Sorvall-TH660 rotor). Pellet, thus obtained, was washed with 1 M NH4Cl and dissolved in a small amount of Buffer G, and stored at −80 °C.
Protein–ribosome co-sedimentation analysis. Purified protein and ribosomes were incubated at 37 °C for 30 min in Buffer E in presence of 2 mM nucleotides (GDP/GMPPMP) and loaded on a 20–43% Sucrose gradient in Buffer F. The tubes were centrifuged at 28,000 RPM for 10 h (Sorvall surespin-630 rotor) and 400 μl fractions collected from the top of the tube were analyzed by SDS–PAGE. Protein was detected by immuno-blotting using anti-His antibody (Santacruz). As above, the presence of 50S/30S in each of the fractions was determined by the presence of 16S rRNA (~1.5 Kb band) and 23S rRNA (~2.9 Kb band). Co-sedimentation experiments with purified 50S and 30S subunits were carried out similarly, except replacing the sucrose gradient to 18–50%.
GTP and ATP hydrolysis assays. GTP hydrolysis assays were carried out in 5 μl reaction volumes containing 20 μM HflX, 50 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM DTT, 5 mM MgCl2, 20 μM GTP, 1 μCi γ[32P] GTP, and were incubated at 37 °C for 60 min. The reaction was stopped by adding 1 μl of 6 M formic acid and centrifuged at 13,000 RPM for 10 min. Five microliters of the sample was spotted on the PEI-coated TLC (Merck), resolved in 1.5 M KH2PO4 (pH 3.4) buffer and subjected to autoradiography to detect the formation of inorganic PO4. Autoradiograms were aligned with the TLC plate and spots corresponding to inorganic PO4 were cored out. The counts (CPM) were determined using a scintillation counter. For ribosome stimulation assays, varying amounts of 50S (0–20 pmoles) were used along with. In the competition assays, GTP hydrolysis (without the ribosome) was carried out using 0–500 μM ATP/AMPPNP/ADP. Similarly, for ATP hydrolysis assays GTP was replaced by ATP and competed with 0–500 μM GTP/GMPPNP/GDP. In competition assays, CPM obtained in absence of competitor was normalized to 100%. Percent activities for the other samples were calculated with respect to this.