My PhD Work:
GTPases are the universally conserved class of regulatory molecules involved in diverse cellular functions such as signal transduction, translation, cytoskeleton formation and intracellular transport. Recent reports shows that bacteria posses 11 universally conserved and essential GTPases (elongation factor G and Tu, initiation factor2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of these indicate that a conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes, suggesting their necessary role either in ribosome biogenesis/assembly or function. EngA is one of these 11 conserved bacterial GTPases which is unique due to presence of two consecutive G-domains, having distinct nucleotide binding and hydrolysis activities, followed by a KH domain at the c-terminus. In the work leading to my thesis, using mutations that allow isolating the activities of the two G-domains, GD1 and GD2, 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 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. The importance of interactions stabilizing the GD1-KH interface was investigated using in vitro EngA-ribosome interaction assays. These studies reveal that formation or disruption of these interactions is necessary to provide nucleotide state specific ribosomal binding. Furthermore, species specific variations at the linker region connecting the two G domains in EngA were found to be important for the ribosome binding function of EngA, as revealed by bioinformatics studies and later verified experimentally. |
Sushil Kumar Tomar. Elucidating a double switch mechanism and inter-domain regulation in a unique ribosome binding bacterial GTPase EngA. Indian Institute of Technology-Kanpur, India-208016. July 2010.
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Publications From PhD work
1) Sushil Kumar Tomar, Neha Dhimole, Moon Chatterji and Balaji Prakash. Distinct GTP/GDP bound states of the tandem G-domains of EngA regulate ribosome binding. Nucleic Acids Research (2009) 37(7)2359-70. Pubmed
2) Sushil Kumar Tomar, Prashant Kumar, Soneya Majumdar, Varun Bhaskar, Prasun Dutta and Balaji Prakash. Extended C-terminus and length of the linker connecting the G-domains are species-specific variations in the EngA family of GTPases. FEBS Open Bio. 2 (2012) 191-195. FEBS Open Bio
Full list of publications
1) Sushil Kumar Tomar, Neha Dhimole, Moon Chatterji and Balaji Prakash. Distinct GTP/GDP bound states of the tandem G-domains of EngA regulate ribosome binding. Nucleic Acids Research (2009) 37(7)2359-70. Pubmed
2) Sushil Kumar Tomar, Prashant Kumar, Soneya Majumdar, Varun Bhaskar, Prasun Dutta and Balaji Prakash. Extended C-terminus and length of the linker connecting the G-domains are species-specific variations in the EngA family of GTPases. FEBS Open Bio. 2 (2012) 191-195. FEBS Open Bio
Full list of publications