Browsing by Subject "Elongation factor tu"

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    Functional role of the noncatalytic domains of elongation factor tu in the interactions with ligands
    (1998) Cetin, R.; Anborgh, P. H.; Cool, R. H.; Parmeggiani, A.
    Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(AM) and EF-Tu(δC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N- terminal domain, they had lost a typical feature of EF-Tu, the ≤ 100 times stronger affinity for GDP than for GTP. EF-Tu(ΔM) and EF-Tu(AC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(ΔC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was ≤90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF- Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(ΔC)·GTP, showing the same kind of effect as on EF-Tu·GTP, but they were little active on EF-Tu(ΔM)·GTP. Like EF-Tu(ΔC)·GTP, EF-Tu(ΔM)·GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.