The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Abstract

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.

Characterization of the Snowy Cotyledon 1 Mutant of Arabidopsis Thaliana: The Impact of Chloroplast Elongation Factor G on Chloroplast Development and Plant Vitality

“…The prokaryotic EF-G, a multidomain GTPase, catalyzes the translocation of the ribosome from the A to the P site on an mRNA transcript (Sharer et al, 1999;Mohr et al, 2000). Mutational analysis of the elongation factor G from E. coli revealed diverse functions of different domains during binding to the ribosomal complex and the translocation step (Borowski et al, 1996;Martemyanov and Gudkov, 1999;Sharer et al, 1999;Mohr et al, 2000). The conserved region adjacent to the GTP-binding effector domain has been shown to be required for the interaction with the 70S ribosome (Sharer et al, 1999).…”

“…Mutational analysis of the elongation factor G from E. coli revealed diverse functions of different domains during binding to the ribosomal complex and the translocation step (Borowski et al, 1996;Martemyanov and Gudkov, 1999;Sharer et al, 1999;Mohr et al, 2000). The conserved region adjacent to the GTP-binding effector domain has been shown to be required for the interaction with the 70S ribosome (Sharer et al, 1999). In the sco1 mutant the mutation results in a replacement of the conserved glycine in position 132 by an arginine residue within this 70S-ribosome-binding region.…”

Etioplast and etio-chloroplast formation under natural conditions: the dark side of chlorophyll biosynthesis in angiosperms

“…A developmental phase regulation is probably in action in the Snowy COtyledon 1 mutant (sco mutant). The SCO gene codes for a chloroplast-localized elongation factor G protein that catalyzes the translation of the ribosome from the A to P site of an mRNA transcript (Sharer et al 1999). At the beginning of the growth (e.g., after 3 days), the cotyledons are Chl-less, but the leaves are not, while later both tissues are fully green (Albrecht et al 2006).…”

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast

“…Site directed mutagenesis has frequently been used to analyse the function of specific amino acids in bacterial EFG [26–29]. To date, there are only a few reports in which this technique has been used to acquire information on the importance of specific amino acids and amino acid motifs for eEF2 function [6,13,30,31].…”