Anatoly T. Gudkov

Crystal structure of a mutant elongation factor G trapped with a GTP analogue

Elongation factor G (EF‐G) is a G protein factor that catalyzes the translocation step in protein synthesis on the ribosome. Its GTP conformation in the absence of the ribosome is currently unknown. We present the structure of a mutant EF‐G (T84A) in complex with the non‐hydrolysable GTP analogue GDPNP. The crystal structure provides a first insight into conformational changes induced in EF‐G by GTP. Comparison of this structure with that of EF‐G in complex with GDP suggests that the GTP and GDP conformations in solution are very similar and that the major contribution to the active GTPase conformation, which is quite different, therefore comes from its interaction with the ribosome.

The L7/L12 ribosomal domain of the ribosome: structural and functional studies

The L7/L12 protein forms a functionally important domain in the ribosome. This domain is involved in interaction with translation factors during protein biosynthesis. The tertiary and quaternary structure of the L7/L12 protein was established as a result of intensive studies in solution and in the ribosome. The conformational changes of L7/L12, the elongation factors Tu and G and other ribosomal proteins were traced by different experimental techniques. These changes occur upon interaction of the ribosome with the elongation factors and depend on GTP hydrolysis in accordance with the functional states of the ribosome.

Direct expression of PCR products in a cell-free transcription/translation system: synthesis of antibacterial peptide cecropin

A simple and effective methodology is proposed for direct expression of PCR‐generated linear DNAs in cell‐free transcription/translation systems without cloning DNA fragments in plasmids. This methodology is realized for the synthesis of the active antibacterial peptide cecropin using the synthetic coding sequence. Possible scientific applications and perspectives of the proposed approach are discussed.

The structure and dynamics of ribosomal protein L12

The protein L12 in bacterial ribosomes is essential for the proper function of a number of factors involved in protein synthesis. The protein is mostly described in terms of a rigid structure despite the repeated observation of high flexibility. This paper gives a review of the structure and flexibility of L12 in relation to its function.

Conformational changes in ribosomes upon interaction with elongation factors.

Conformational changes in the ribosomes upon interaction with EF-Tu were studied by limited proteolysis with a set of proteases. The main results are: (1) The cleavage rate of S1 protein strongly depends on the cooperative effect of poly(U) and tRNA: (2) The conformation of L7/L12 proteins is modulated by interaction of elongation factors with the ribosome and depends on hydrolysis of GTP; (3) The sensitivity of some ribosomal proteins (S6, S7, S18, S19, L9, L16, L19, and L27) to proteases changes upon binding of EF-Tu and depends on the ribosome functional state in accordance with GTP hydrolysis. Most of these proteins are located far from the factor-binding center of the ribosome. The possible mechanism of conformational changes is discussed.

Domain IV of elongation factor G from Thermus thermophilus is strictly required for translocation

Two truncated variants of elongation factor G from Thermus thermophilus with deletion of its domain IV have been constructed and the mutated genes were expressed in Escherichia coli. The truncated factors were produced in a soluble form and retained a high thermostability. It was demonstrated that mutated factors possessed (1) a reduced affinity to the ribosomes with an uncleavable GTP analog and (2) a specific ribosome‐dependent GTPase activity. At the same time, in contrast to the wild‐type elongation factor G, they were incapable to promote translocation. The conclusions are drawn that (1) domain IV is not involved in the GTPase activity of elongation factor G, (2) it contributes to the binding of elongation factor G with the ribosome and (3) is strictly required for translocation. These results suggest that domain IV might be directly involved in translocation and GTPase activity of the factor is not directly coupled with translocation.

Conformational independence of N- and C-domains in ribosomal protein L7/L12 and in the complex with protein L10

Isolated N‐ (1–37) and C‐terminal (47–120) fragments of L7 protein, and pentameric (L7)4L10 complex were studied by NMR spectroscopy in solution. The results indicate that the dimer state of the 1–37 fragment with a helical hairpin conformation is identical to the N‐terminal structure of the intact L7 dimer. The C‐terminal domain of the L7 protein does not participate in (L7)4L10 complex formation. The overall motions of the L7 C‐domains are essentially independent both in the L7 dimer and in the (L7)4L10 complex. Conformational motions on a millisecond time scale are detected in the (L7)4L10 complex. The possible relevance of these motions to the biological function of L7/L12 is discussed.

Topology of the secondary structure elements of ribosomal protein L7/L12 from E. coli in solution

Topology of the secondary structure elements of ribosomal protein L7/L12 has been studied. The sequential assignment was obtained for main and side chain resonances. This allows the overall secondary structure to be described. The results of high resolution NMR studies show that dimer of the ribosomal protein L7/L12 from Escherichia coli has a parallel (head‐to‐head) orientation of subunits, and N‐terminal domain (NTD, residues Ser1‐Ser33) has no contracts with the C‐terminal domain (CTD, residues Lys51‐Lys120). The NMR data for CTD are in line with crystallographic structure. The flexible interdomain (hinge) region (residues Ala34‐Glu50) has an unordered structure, the Pro44 forming both cis and trans peptide bonds. Due to the conformational exchange the intensities of the peaks from the NTD are low. The conformation of the NTD, which is responsible for the formation of the L7/L12 dimer, is α‐helical hairpin. The NTD dimer forms an antiparallel four‐α‐helix bundle.

Domain III of Elongation Factor G from Thermus thermophilus Is Essential for Induction of GTP Hydrolysis on the Ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 103-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G·GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.

An intact conformation at the tip of elongation factor G domain IV is functionally important.

Three variants of Thermus thermophilus EF‐G with mutations in the loop at the distal end of its domain IV were obtained. The replacement of His‐573 by Ala and double mutation H573A/D576A did not influence the functional activity of EF‐G. On the other hand, the insertion of six amino acids into the loop between residues Asp‐576 and Ser‐577 reduced the translocational activity of EF‐G markedly, while its GTPase activity was not affected. It is concluded that the native conformation of the loop is important for the factor‐promoted translocation in the ribosome. The functional importance of the entire EF‐G domain IV is discussed.