Several GTPases participate in bacterial protein biosynthesis. Initiation factor 2 controls the formation of the ribosomal initiation complex and places initiator fMet-tRNAfMet in the ribosomal P-site. Elongation factors Tu and G are responsible for codon-specific binding of the aminoacyl-tRNA to the A-site, and peptidyl-tRNA to the P-site, respectively, during the elongation phase of protein biosynthesis. Release factor 3, a GTPase which is not ubiquitous, is involved in termination and release of the nascent polypeptide. Other translation factors, including initiation factors 1 and 3, elongation factor Ts, release factors 1 and 2, and ribosomal release factor do not belong to the family of GTP/GDP binding proteins. The guanosine nucleotide binding domains of the GTPases involved in translation are structurally related to the Galpha subunit of heterotrimeric G proteins and to the proteins of the Ras family. We have identified and sequenced all genes coding for translation factors in the extreme thermophile Thermus thermophilus. The proteins were overproduced in Escherichia coli, purified, biochemically characterised and used for crystallisation and structural analysis. Further biochemical investigations were aimed at gaining insight into the molecular mechanism underlying the regulation of the GTPase activity of the translation factors, and to elucidate the role of their ribosomal binding sites in this process.
The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide-and Mg 2؉ -free protein has been investigated. The GTPase superfamily of proteins, known more commonly as G-proteins, are ubiquitous in cellular systems and serve as key regulatory molecules catalyzing the hydrolysis of the ,␥-phosphate bond in GTP (1-4). For most G-proteins this process yields a tightly bound enzyme-product complex requiring the action of a nucleotide exchange factor to kinetically facilitate exchange of the GTP for GDP in the active site. Nucleotide binding and hydrolysis are key to regulating the activity of these enzymes, and both of these steps occur in the presence of the divalent metal ion Mg 2ϩ as the naturally occurring cofactor in the cell. However, in EF-Tu 1 function it has not been possible hitherto to establish whether there is a differential role of the divalent metal ion in nucleotide hydrolysis as opposed to nucleotide binding.In all G-proteins defined hitherto by x-ray crystallographic studies, the substrate analog guanosine 5Ј-(,␥-imido)-triphosphate is bound as a complex with Mg 2ϩ in which the divalent metal ion is coordinated to oxygen atoms from the -phosphate and ␥-phosphate groups, and the guanine and divalent metal ion binding sites appear to be tightly coupled (5, 6). For ras (7), for elongation factor Tu from both thermophilic bacteria (8 -11) and Escherichia coli (12, 13), and for G␣-subunits of heterotrimeric G-proteins (14, 15), the dissociation constants for release of protein-bound nucleotide measured in the presence of Mg 2ϩ indicate differential binding affinities for GDP and GTP by at least an order of magnitude. Binding affinities for both nucleotides are often measured kinetically by ligand displacement because G-proteins are less stable when prepared in their nucleotide-free form. Because nucleotide separation is necessary for complete removal of Mg 2ϩ from the protein, the less stable, nucleotide-free form of G-proteins complicates efforts to determine whether the divalent metal ion has differential roles in nucleotide hydrolysis and nucleotide binding.In contrast to the structural instability of elongation factor Tu of mesophiles such as E. coli, the homologous thermostable protein in its nucleotide-free form is less prone to inactivation, and comparison of the kinetics of nucleotide binding shows it to be identical to EF-Tu isolated as the GDP-bound complex from the cytosol (11,16). This characteristic has allowed application of efficient biochemical purification methods for preparation of the nucleotide-free elongation factor from Thermus thermophilus (16,17) and Bacillus stearothermophilus (18, 19) for x-ray structure analysis of the complex formed with an inhibitor analog of GTP in the active site (5, 6). Isolation of nucleotidefree elongation factor Tu (17-20) and p21 ras (7) has also allowed comp...
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