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

Abstract

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.z 1998 Federation of European Biochemical Societies.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation

“…Although EF-G accelerates translocation by 10 3 -fold to 10 4 -fold more than the A-site antibiotics (Rodnina et al 1997), it may operate through an analogous mechanism, serving as a pawl in a Brownian ratchet mechanism (Frank and Gonzalez 2010;Ratje et al 2010). Indeed, in cryo-EM reconstructions of EF-G-ribosome complexes (Valle et al 2003;Ratje et al 2010), as well as in a recent crystal structure of EF-G bound to a nonrotated, classical-state ribosome (Gao et al 2009), domain IV of EF-G, which has been shown to be essential for catalysis of translocation (Rodnina et al 1997;Martemyanov et al 1998), overlaps with the A site of the small ribosomal subunit. Thus, one role of domain IV of EF-G could be to prevent the return of tRNA back into the 30S A site during translocation.…”

Ribosomal Protection Proteins and Their Mechanism ofTetracyclineResistance

“…Furthermore, a comparison of the EF-G and Tet(O) ribosomal contacts indicates that they differ primarily in the vicinity of domain IV (Table 1), where EF-G contacts H69 of the 23S rRNA (21,45) and Tet(O) interacts with h18/34 of the 16S rRNA (45). This is significant, as domain IV in EF-G has been implicated as an important determinant for promoting translocation of the tRNAs (30,31,42). In this case, these differences in domain IV may serve to distinguish Tet(O) and EF-G with respect to their activities; namely, domain IV of EF-G more intimately overlaps with the A site-bound tRNA, an idea that is consistent with the role of domain IV of EF-G in translocation.…”

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

“…Earlier it was shown that dimeric cecropin synthesized in the plasmid-programmed cell-free system is not active [11], at least at concentrations where monomeric cecropin displays activity. Since the active cecropin molecule does not contain methionine, the multimeric polypeptide produced in the cellfree system could be subjected to CNBr cleavage.…”

“…A set of multimeric cecropin genes was obtained by the modified splicing overlap extension technique [11] with the use of primers Prl and Pr2. A conceptually similar procedure to polymerize DNA fragments encoding decapeptides was published recently [12].…”

“…The long primer (megaprimer) containing the T7 promoter and the ribosome binding site (RBS) with the slO leader and Shine-Dalgarno sequences was generated by PCR from plasmid pET21d(+) [7] with the use of primers Pr3 and Pr4. The megaprimer and primer Pr5 were added to the products of the first PCR, containing up to seven repeats of the cecropin coding sequence [11], and the next PCR round resulted in production of the linear DNA with necessary elements for direct expression in a cell-free system (Fig. 1). …”