2362 Nucleic Acids Research, 2009, Vol. 37, No. 7 Figure 1. EngA interacts with rRNA and ribosomal subunits. (A) 16S rRNA and 23S rRNA co-elute with EngA. The E. coli BL21 (DE3) cells overexpressing EngA were lysed in RNAse-free environment and EngA was puriﬁed as described in Materials and methods section. Lane 1 shows the rRNA following phenol–chloroform extraction of a fraction containing EngA. The identity of the rRNA was conﬁrmed by RT-PCR using speciﬁc primers for 16S and 23S rRNA. Lanes 2 and 3 show the DNA fragments ampliﬁed from the co-eluted rRNA, using 23S rRNA and 16S rRNA primers, respectively. DNA ladder (Fermentas, Mass ruler SM0403) loaded in lane 4, suggest that the sizes of the ampliﬁed DNA fragments correspond to these rRNAs. (B) The ability of GST–EngA and EngA to hydrolyse GTP was analysed by using malachite green assay (see Materials and methods section). Bar plots indicate that the amount (%) of GTP hydrolysed by GST-tagged and untagged EngA are similar. (C) EngA–ribosome co-fractionation experiments were conducted using the same cells. Cells lysed either in absence of nucleotides or in presence of GDP or GMPPNP, were sedimented through a 15–43% sucrose gradient at 28 000 RPM (Sorvall Sure-spin 630 rotor) and fractions were collected while monitoring A254. A proﬁle indicating the diﬀerent ribosomal subunits is shown. (D) The fractions were analysed on a 12% SDS–PAGE and followed by immuno-blotting with anti-GST antibody. EngA does not associate with ribosomes (D2) in absence of nucleotides (Apo) or (D3) in presence of GDP. However, it associates with 50S ribosomal subunit in the presence of GMPPNP at (D4) 50 mM, (D5) 150 mM and also at (D6) 350 mM NH4Cl. The inﬂuence of the GST-tag on these interactions is negated in D1, where GST (lacking EngA) was used as a negative control in these experiments. upon overexpression. The GST fused EngA proteins produced in pGEX vector, increased protein solubility signiﬁcantly (see Materials and methods section). Hence, GST–EngA was used for further experimentation. The GST–EngA overexpressing (E. coli) cells were lysed in an RNase-free environment and the protein (GST– EngA) was puriﬁed by GSTrap aﬃnity puriﬁcation. When this was subjected to size-exclusion chromatogra- phy experiments, it eluted in the void volume fractions (data not shown). A phenol–chloroform extraction of the fractions containing GST–EngA suggests that both 23S rRNA and 16S rRNA co-elute with the protein (Figure 1A, lane 1). The identity of the rRNAs was ver- iﬁed using RT-PCR employing primers speciﬁc for 16S rRNA and 23S rRNA and the co-eluted rRNA as a tem- plate. In Figure 1A, lane 2 shows the DNA ampliﬁed using 23S rRNA primers and lane 3 shows the DNA ampliﬁed using 16S rRNA primers. Size of the ampliﬁed DNA fragments was found to be 2.9 and 1.5 kb, respec- tively, in accordance with the sizes of 23S and 16S rRNAs. Figure 1A thus reveals that not only 23S rRNA co-elutes with the protein, as we had anticipated, but also the 16S rRNA. In agreement with this observation, Hwang and Innoye (15) also found that der-depleted cells accumulate 16S and 23S rRNA precursors. EngA belongs to the family of universally conserved bacterial GTPases, of which some like Era have been shown to bind the 16S rRNA, but not 23S rRNA (24). Most members of this family are now known to interact with ribosomal subunits in a speciﬁc nucleotide-bound state of the protein (4–15). These observations, together with the co-elution of 16S and 23S rRNA, suggested that EngA would bind the 30S and 50S ribosomal subunits. To verify ribosome binding, co-fractionation experiments were designed using cells overexpressing GST–EngA. Prior to this, to rule out any additional eﬀect the bulky GST-tag may have on the biochemical behaviour of the protein, we analysed the enzymatic activity, i.e. the ability to hydrolyse GTP, of GST–EngA and EngA using the malachite green assay (20). Figure 1B shows that GTP hydrolysis by both GST-tagged and untagged EngA are similar. The co-fractionation experiments designed to verify interactions of EngA with ribosomal subunits, were