Nucleic Acids Research Advance Access originally published online on February 26, 2009
Nucleic Acids Research 2009 37(7):2359-2370; doi:10.1093/nar/gkp107
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Nucleic Acids Research, 2009, Vol. 37, No. 7 2359-2370
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Distinct GDP/GTP bound states of the tandem G-domains of EngA regulate ribosome binding
Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India
*To whom correspondence should be addressed. Tel: +91 512 259 4013; Fax: +91 512 259 4010; Email: bprakash{at}iitk.ac.in
Received January 10, 2009. Revised February 9, 2009. Accepted February 9, 2009.
EngA, a unique GTPase containing a KH-domain preceded by two consecutive G-domains, displays distinct nucleotide binding and hydrolysis activities. So far, Escherichia coli EngA is reported to bind the 50S ribosomal subunit in the guanosine-5'-trihosphate (GTP) bound state. Here, for the first time, using mutations that allow isolating the activities of the two G-domains, GD1 and GD2, we show that apart from 50S, EngA also binds the 30S and 70S subunits. We identify that the key requirement for any EngA–ribosome association is GTP binding to GD2. In this state, EngA displays a weak 50S association, which is further stabilized when GD1 too binds GTP. Exchanging bound GTP with guanosine-5'-diphosphate (GDP), at GD1, results in interactions with 50S, 30S and 70S. Therefore, it appears that GD1 employs GTP hydrolysis as a means to regulate the differential specificity of EngA to either 50S alone or to 50S, 30S and 70S subunits. Furthermore, using constructs lacking either GD1 or both GD1 and GD2, we infer that GD1, when bound to GTP and GDP, adopts distinct conformations to mask or unmask the 30S binding site on EngA. Our results suggest a model where distinct nucleotide-bound states of the two G-domains regulate formation of specific EngA–ribosome complexes.