(40 mM) over mant-XDP, resulting in a GDP:mant-XDP ratio of 100:1. Malachite green assay GTP hydrolysis by GST–EngA and EngA was measured in a reaction buﬀer. Equimolar (500 nM) amount of pro- tein was incubated with 500 mM of GTP in Buﬀer F for 25 min at room temperature. Released phosphate was measured by using a colorimetric malachite green assay as described in (20). A separate reaction was setup without any protein to estimate the amount of GTP hydrolysed during the time of the reaction. This was negated from the aforesaid measurements to estimate the amount of inor- ganic phosphate released due to the GTP hydrolysis by the proteins. All the reactions were setup in triplicates. RNA association with EngA GST–EngA was puriﬁed using aﬃnity chromatography. The complete experiment was carried out in an RNAse- free environment. Cells were lysed in Buﬀer A containing 1% Tween-20, 5 mM MgCl2, 50 mM NaCl, 0.01 mM AEBSF and 1 mg/ml lysozyme by repeated freeze-thaw cycles. Lysates, treated with DNAse, were centrifuged at 30 000 g for 1 h at 48C. Supernatant was loaded on a 5 ml GSTrap aﬃnity column (Amersham), pre-equilibrated with Buﬀer A. Unbound protein was washed (with Buﬀer A) and protein was eluted in the same buﬀer containing 10 mM glutathione. Fractions containing protein were analysed by size exclusion chromatography on HiLoad 16/60 Superdex 200 column (Amersham). Co-eluted rRNA was recovered from the protein by phenol–chloroform extraction and was analysed on 1% agarose–formaldehyde gel. The identity of the rRNA was further conﬁrmed by RT-PCR using AMV reverse tran- scriptase enzyme (Genei) using speciﬁc primers for 16S (forward—AAATTGAAGAGTTTGATCATGGCTCA GATT and reverse—TAAGGAGGTGATCCAACCGC AGGTTCCCC) and 23S rRNA (forward—GGTTAAG CGACTAAGCGTACACGGGTGGATG and reverse— AAGGTTAAGCCTCACGGTTCATTAGTACCG). Ribosome profiling Wild type (wt) and mutants of EngA were expressed in E. coli BL21 (DE3) as described above with the following modiﬁcation. Before harvesting the cells, chloramphenicol was added to a ﬁnal concentration of 100 mg/ml on ice for 10 min to trap the polysomes. Cells, harvested and washed with Buﬀer C (50 mM Tris–HCl pH 7.5, 10 mM MgCl2 and 50 mM NH4Cl), were lysed in an RNAse-free envir- onment, in the same buﬀer with appropriate nucleotides (xanthine-5-[b,g-imido]triphosphate (XMPPNP) (Jena Bioscience), GDP or/and GMPPNP (Sigma)) at 1 mM concentrations and with 50–350 mM NH4Cl, as the case may be. The lysate was centrifuged at 18 000 g for 30 min. The ribosomal subunits (A254 10 units) were resolved on a 17 ml continuous sucrose gradient (15–43%) in Buﬀer D (20 mM Tris–HCl pH 7.5, 10 mM MgCl2 and 50–350 mM NH4Cl). Gradient was subjected to ultra centrifugation at 28 000 RPM (Sorvall Sure-Spin 630 rotor) for 10 h. Thirty equal fractions (seven drops each) were collected from top Nucleic Acids Research, 2009, Vol. 37, No. 7 2361 to bottom using ISCO Gradient Maker, while monitoring A254. Fractions 5–30 (corresponding to lanes 1–26 in all Figures 1, 2 and 3) were resolved on SDS–PAGE followed by immuno-blotting using anti-GST antibodies (Santa Cruz Biotechnology) as all constructs of EngA described here contain an N-terminal GST fusion tag. Ribosome binding experiments using purified components For Figure 3C, 100 pmol protein(s) were incubated with ﬁve A254 crude ribosomes in presence of appropriate nucleotides (1 mM) in Buﬀer C. The reaction mixtures were then resolved on a manually prepared 17 ml step gra- dient of sucrose in Buﬀer D. The gradient was subjected to ultra centrifugation at 28 000 RPM (Sorvall Sure-Spin 630 rotor) for 10 h. Thirty equal fractions (seven drops each) were collected from top to bottom using ISCO Gradient Maker, while monitoring A254. RNA was isolated from the fractions and was analysed by formaldehyde–agarose gel (1.5% agarose) for the presence of 16S and 23S rRNA. Based on this, peak fractions containing 30S, 50S and 70S subunits were analysed on a SDS–PAGE followed by immuno-blotting using anti-GST antibodies. For Figure 4, ribosomes were puriﬁed from B. subtilis strain using the protocol employed for ribosome proﬁling. Fractions were analysed for the presence of various ribo- somal subunits, as above. To generate a pure sample of the ribosomal subunits, only the peak fractions corre- sponding to the various ribosomal subunits were pooled, concentrated and were snap frozen in liquid nitrogen and stored at 808C for further use. The 100 pmol protein were incubated with ﬁve A254 of the puriﬁed ribosomal subunits in presence of appropriate nucleotides (1 mM) in Buﬀer C. The reaction mixtures were loaded on a 4 ml cushion of 25% sucrose in Buﬀer D and were then subjected to ultra centrifugation at 40 000 RPM (TH 660 rotor) for 2 h. The supernatant and pellet fractions were analysed for the presence of the protein by immuno-blotting using anti-His antibodies. Structural superposition Structures of B. subtilis YphC (PDB:2HJG) and T. maritima Der (PDB:1MKY) were superimposed using Coot (21). Figures 4A and 5C were prepared using CHIMERA (22). RESULTS EngA binds the 50S subunit in the GTP-bound state The E. coli RrmJ, a heat shock controlled rRNA methyl- transferase modiﬁes the 23S rRNA in intact 50S ribosomal subunits. The rrmJ mutants lead to signiﬁcant accu- mulation of ribosomal subunits at the expense of func- tional 70S particles, which are restored to normal levels upon overexpressing EngA (23). This suggests a functional relationship between EngA and 23S rRNA or the 50S subunit. We ﬁrst wanted to examine if EngA binds 23S rRNA, as RrmJ does. EngA, cloned in pQE2 and pET28A vectors to produce His6-tagged protein, led to highly insoluble aggregates,