The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fussA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42°C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within Jis4. ThefusA4100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fus4101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of 0-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo.These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.In bacteria, the elongation steps of protein synthesis require the sequential action of two different elongation factors, EF-G and EF-Tu. EF-Tu is required to deliver the correct aminoacyltRNA to the A site on the ribosome and is, therefore, intimately involved in proofreading (for a review, see reference 27). EF-G catalyzes ribosome translocation on mRNA through a mechanism that somehow is enhanced by hydrolysis of GTP by EF-G (for a review, see reference 15). The movement of the ribosome is an intricate mechanical process that remains relatively poorly understood. Previous biochemical investigations have been aimed at defining the sites at which EF-G binds to ribosomal protein and rRNA. From these data, it is apparent that EF-G binds to the ribosome at the base of the L7/L12 stalk and is closely associated with proteins L10 and Lii among others (for a review, see reference 26). Crosslinking and RNA protection experiments provide convincing evidence that part of the binding site is also composed of the 23S rRNA near nucleotides 2660 and 1067 (21, 24). In addition, in vitro experiments have shoWn that the L7/L12 protein dimer is essential for EF-G binding and function (10,13,22). Little is known about the residues in EF-G which participate in these interactions. In particular, there have been relatively few investigations that use bacterial genetics to select for informative mutations in fusA (the Escherichia coli gene that encodes EF-G). One of the early studies used the incorporation of tritiated precursors to induce death by suicide (25). A temperatur...

Regulation of phosphatidate phosphatase (EC 3.1.34) activity was examined in Saccharomyces cerevisiae cells supplemented with phospholipid precursors. Addition of inositol to the growth medium of wild-type cells resulted in a twofold increase in phosphatidate phosphatase activity. The increase in phosphatidate phosphatase activity was not due to soluble effector molecules, and inositol did not have a direct effect on enzyme activity. The phosphatidate phosphatase activity associated with the mitochondrial, microsoinal, and cytosolic fractions of the cell was regulated by inositol in the same manner. Cells supplemented with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared with unsupplemented cells. Serine, ethanolamine, and choline did not significantly affect the phosphatidate phosphatase activity of cells grown in the absence or presence of inositol. Enzyme activity was not regulated in inositol biosynthesis regulatory mutants, suggesting that regulation by inositol is coupled to regulation of inositol biosynthesis. Phosphatidate phosphatase activity was pleiotropically expressed in structural gene mutants defective in phospholipid biosynthesis. These results suggested that phosphatidate phosphatase was regulated by inositol at a genetic level.Phosphatidate (PA) is an important branch point intermediate for the synthesis of phospholipids, diacylglycerols, and triacylglycerols in the unicellular eucaryote Saccharomyces cerevisiae (19). In the primary pathway of phospholipid biosynthesis, phosphatidylcholine is derived from PA via the reaction sequence PA -+ CDP-diacylglycerol phosphatidylserine -> phosphatidylethanolamine -* phosphatidylmonomethylethanolamine -* phosphatidyldimethylethanolamine -> phosphatidylcholine (19). Phosphatidylinositol, phosphatidylglycerol, and cardiolipin are also derived from CDP-diacylglycerol (19). Diacylglycerols and triacylglycerols are derived from PA via the reaction sequence PA -* diacylglycerol --triacylglycerol (19). The diacylglycerol synthesized from PA can also be used for synthesis of phosphatidylethanolamine and phosphatidylcholine by the auxiliary CDP-ethanolamine-and CDP-choline-based pathways, respectively (28), when synthesis of phosphatidylserine is blocked and cells are supplemented with ethanolamine and choline (2,32,36). Addition of.ethanolamine or choline to inositol-containing growth medium leads to repression of the enzymes (CDPdiacylglycerol synthase [24], phosphatidylserine,synthase [3,7,30,37], phosphatidyiserine decarboxylase [8], and the phospholipid N-methyltransferases [8,30,42,43,45,46]) in the primary pathway for phosphatidylcholine biosynthesis. Repression of these enzymes by ethanolamine and choline is absolutely dependent on inositol (3,24,30,37,45,46). Inositol alone partially represses these enzymes (3,24,30,37,45,46). The enzyme responsible for synthesis of phosphatidylinositol from CDP-diacylglycerol (phosphatidylinositol synthase) is not repressed by phospholipid precursors (15,30). However, the enzyme resp...

Era is an Escherichia coli GTPase that is essential for cell viability and is peripherally associated with the cytoplasmic membrane. Both immunoelectron microscopy and subcellular-fractionation experiments have shown that Era is present in cytoplasmic as well as membrane-associated pools. These data led to speculation that the mechanism of action of Era may require cycling between membrane and cytoplasmic sites. In order to investigate this possibility, an in vitro binding assay was developed to characterize the binding of Era to membrane fractions. Competition and saturation binding experiments suggest that a site that is specific for Era and capable of binding up to 5 ng of Era per microgram of membrane protein is present in membrane preparations. The binding curve is complex, indicating that multiple equilibria describe the interaction. The binding of Era to this putative receptor is dependent on guanine nucleotides; binding cannot be measured in the absence of nucleotide, and neither ATP nor UTP can substitute. Subfractionation of cell walls showed that the guanine nucleotide-dependent binding site was present in fractions enriched in cytoplasmic membrane. These data provide evidence that Era may be involved in a GTPase-receptor-coupled membrane-signaling pathway that is essential for growth in E. coli.

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