2360 Nucleic Acids Research, 2009, Vol. 37, No. 7 its GTP and GDP-bound forms. By comparing structures of Der (16) and YphC, Muench et al. (17) hypothesize that the GTP-bound state of GD1 exposes a positively charged RNA-binding region on the surface of KH-domain, which is masked due to the large movement of GD1. Schaefer et al. (14) indicate that the ribosome exits YphC in its GDP-bound state. On the whole, these studies indicate an importance for the two G-domains of EngA in binding the ribosomal subunits. Hitherto, EngA was shown to bind the 50S ribosomal subunit. Here, for the ﬁrst time, we show that E. coli EngA not only binds the 50S but also 30S and 70S. We identify two distinct ribosome-bound states of EngA—one where it binds 50S alone and another where it binds 50S, 30S and 70S. We associate this diﬀerential binding to the diﬀerent nucleotide occupancy states of GD1 and GD2, based on co-fractionation experiments with ribosomal subunits using EngA mutants, which either disable nucleotide binding or change speciﬁcity from guanine to xanthine nucleotides (18). Surprisingly in a very recent report BipA, a highly conserved prokaryotic GTPase, was also shown to bind 70S and 30S subunits, although the latter is achieved in presence of ppGpp, an alarmone synthesized during stress. The 70S binding to BipA is realized in pres- ence of GMPPNP (19). In contrast, here we show that EngA achieves the two distinct ribosome-bound states by varying the GTP or GDP-bound states of the two G-domains. MATERIALS AND METHODS Plasmids The gene coding for EngA (503 amino acids) was ampli- ﬁed using forward (CTAGCTAGCGTGCGTTGTCTGA TGAT) and reverse (CCGCTCGAGTTATTTATTTTTC TTGATGTG) primers from E. coli k12 genomic DNA, using Pfu DNA polymerase (Fermentas). Amplicon was digested with NheI/XhoI and cloned into corresponding sites in modiﬁed pGEX expression vector. Two truncated constructs of EngA, GD1–EngA (213–503 amino acids) and GD1– GD2EngA (386–503 amino acids), were generated in a similar way using forward primers (CGCGCTAGCAGTCTGCCGATCAAACTGC) and (CGCGCTAGCAGCTCCACCCGTCGTGTGGG), res- pectively. His-YphC construct was prepared similarly by amplifying the gene coding for YphC using forward (CCG CATATGATGGGTAAACCTGTCGTA) and reverse primers (CGCGCGGCCGCTTATTTTCTAGCTCTC) from B. subtilis genomic DNA, using Pfu DNA polymer- ase (Fermentas). Amplicon was digested with NdeI/NotI and cloned into corresponding sites in pQE2 expression vector. The clones were conﬁrmed by DNA sequencing. Site-directed mutagenesis All the mutants of EngA i.e. K28A, K228A, D134N and D337N and YphC-Y134A/D297N used in this study were generated by overlapping PCR technique using EngA full length as a template. Primers were designed with the desired change in the codon sequence. Single mutants K28A, K228A, D134N and D337N were generated using primers (GGGCGCCCTAACGTAGGAGCATCC ACGTTA), (CCGAACGTAGGTGCGTCTACACTCA CT), (GTGGCAAACAAAACTAACGGTCTGGATC CC) and (GGTGAATAAGTGGAATGGCTTAAGTC AGGAAGTG), respectively. His-YphC double mutant Y134A/D297N was also generated similarly using primers (GCGAATATTTATGATTTTGCATCGCTAGGCTTT GGC, GTAAACAAATGGAATGCTGTTGACAAAG AT) and His-YphC full length as a template. All muta- tions were conﬁrmed by DNA sequencing. Protein expression and purification All constructs and mutants of EngA were expressed in E. coli BL21 (DE3). Cells were grown up to OD600 0.6– 0.8 at 378C in presence of 100 mg/ml of ampicillin in Luria Bertani media (Himedia) and were induced with 50 mM isopropyl-b-D-1-thiogalacto-pyranoside (IPTG) (Sigma) at 188C for 10 h. Cells were harvested by centrifugation at 4000 g for 5 min at 48C and washed with Buﬀer A (50 mM sodium-phosphate pH 7.4 and 100 mM NaCl). Similarly His-YphC and its double mutant were expressed in E. coli DH5 cells. Cells were grown similarly, but induced with 250 mM IPTG (Sigma) at 308C for 5 h. Cells were harvested by centrifugation at 4000 g for 5 min at 48C and washed with Buﬀer B (50 mM Tris– HCl pH 8.0 and 250 mM NaCl). To purify YphC protein, cells were lysed in Buﬀer B containing 5 mM MgCl2, 350 mM NaCl, 0.01 mM AEBSF [4-(2-Aminoethyl) benze- nesulfonyl ﬂuoride hydrochloride] and 1 mg/ml lysozyme by repeated freeze-thaw cycles. Lysates, treated with DNAse and RNAse, were centrifuged at 30 000 g for 1 h at 48C. Supernatant was loaded on a 5 ml His-trap aﬃnity column (Amersham), pre-equilibrated with Buﬀer B. Unbound protein was washed (with Buﬀer B) and protein was eluted in the same buﬀer using 0–500 mM gradient of imidazole. Fractions containing the protein were analysed by size exclusion chromatography on sephacryl 200 column (Amersham). GST–EngA was puriﬁed as described below (‘RNA association with EngA’), except the use of RNAase and 450 mM NaCl during cell lysis. Fluorescent nucleotide binding assays Fluorescent nucleotide binding studies were carried out using LS 55 Fluorescence Spectrometer (PerkinElmer Life Sciences). The N-methyl-30-O-anthranoyl (mant) group attached to the nucleotides was monitored with an excitation wavelength of 355 nm (slit width of 5 nm) and emission wavelength of 400–600 nm (slit width of 10 nm). Emission proﬁles of mant-nucleotides were gener- ated from 0.4 mM free mant-nucleotides in Buﬀer F con- taining 50 mM Tris–HCl (pH 8.0), 400 mM KCl, 150 mM NaCl and 2 mM MgCl2. Protein mant-nucleotide com- plexes were generated by incubating 8.0 mM of the respec- tive proteins with 0.4 mM mant-nucleotides in Buﬀer F at room temperature. The binding of D134N, D337N and Y134A/D297N mutants for xanthine nucleotides was studied by measuring mant-XDP binding and the speciﬁ- city was assessed in presence of a 100 fold excess of GDP