Nucleic Acids Research, 2009, Vol. 37, No. 7 2367 Figure 4. GD1–KH interactions are important for ribosome binding. (A) A structural superposition of EngA homologues, Der (PDB:1MKY) and YphC (PDB:2HJG), showing a 60 A˚ movement of GD1 between the GTP- and GDP-bound forms. Tyr134 of YphC, a key residue stabilizing the GD1–KH interface is shown by an arrow. (B) In vitro interactions with the puriﬁed 30S, 50S and 70S ribosomal subunits, as described in Materials and methods section were carried out using wt-His-YphC and the double mutant (Y134A, D297N). Immuno-blots using anti-His antibodies, for the supernatant and pellet fractions are shown. The wt-His-YphC did not display any interactions in presence of GMPPNP whereas the YphC double mutant restored interactions with the 50S in presence of GMPPNP and XMPPNP. Interaction of the double mutant with 30S, 50S and 70S was also found in presence of GDP and XMPPNP. Fluorescent nucleotide binding assays were carried out with wt-His-YphC and the double mutant (Y134A, D297N) in presence of mant-XDP and mant-GDP. Emission spectra conﬁrms binding of mant-GDP to wt-His-YphC and both mant-GDP and mant-XDP to the double mutant. mutant restored interactions with the 50S in EngA[GTP:GTP] state and also with 30S, 50S and 70S in the EngA[GDP:GTP] state (Figure 4B). These results are in accordance with the in vivo interactions displayed by GST–EngA (Figures 1D and 3A). The mant-XDP and mant-GDP binding to wt-HisYphC and the double mutant were veriﬁed, as shown in Figure 4B. DISCUSSION EngA is essential for bacterial cell growth. Cells depleted with engA show slower growth patterns (29). Current reports show that it interacts with the ribosome (13,15) and participates in its biogenesis (14). EngA is particularly interesting and distinctive due to the two tandem G-domains, GD1 and GD2, it possesses, apart from the KH-domain. The results provided here demonstrate that by the use of distinct nucleotide-bound states of the two G-domains, EngA regulates ribosome binding. A previous report has shown that a mutant of rrmJ, an rRNA methylase, displays an increase in the level of unassembled 30S and 50S subunits and a decrease in 70S. This eﬀect was restored upon overexpressing EngA or Obg (23), indicating their role in ribosome assembly (formation of 70S). Apart from this, Obg and EngA are known to be involved in ribosome biogenesis (7,14) and Obg is also known to bind both 30S and 50S subunits (7). Similarly, we found EngA to co-elute with both 23S and 16S rRNA (Figure 1A), although RrmJ only binds and modiﬁes the 23S rRNA. However, like other investigators (13,15), we too found EngA to interact only with the 50S subunit in a GTP dependent manner, but not with the 30S (Figure 1D, D4–D6). 50S interaction accounts for the co-elution of 23S rRNA, while 16S rRNA