RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex.

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.

Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid.

Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.

Cell-free production of biologically active polypeptides: application to the synthesis of antibacterial peptide cecropin.

An approach to preparative production of polypeptides, including uneasily testable, degradable, and toxic ones, is proposed on the basis of in vitro expression systems of last generation, such as continuous-exchange cell-free and continuous-flow cell-free transcription-translation systems. The approach implies that a polypeptide of interest is synthesized as a fusion protein with the polypeptide linked to green fluorescent protein (GFP) through a cleavable spacer. The GFP moiety provides direct visualization and quantitative monitoring of the polypeptide synthesis, as well as solubility and stability of the product. The synthesis of functionally active antibacterial polypeptide cecropin P1 (31 amino acid residues) has been demonstrated.

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 10(3)-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.

Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system.

The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.

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.

Increased functional activity of elongation factor G with G16V mutation in the GTP-binding domain.

Oligonucleotide-directed mutagenesis was used to obtain elongation factor G from Thermus thermophilus with the G16V mutation in its GTP-binding domain. Functional studies of the mutated protein and elongation factor G from E. coli were carried out. The data revealed that the G16V mutant retains high thermostability, has an increased ribosome-dependent GTPase activity, and its translation activity in cell-free translation system is equal to that of the factor G from E. coli. The mutated protein with an uncleavable GTP analog also has an increased affinity to the ribosomes.

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.

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.