The GTPases comprise a protein superfamily of highly conserved molecular switches adapted to many diverse functions. These proteins are found in all domains of life and often perform essential roles in fundamental cellular processes. Analysis of data from genome sequencing projects demonstrates that bacteria possess a core of 11 universally conserved GTPases (elongation factor G and Tu, initiation factor 2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of GTPases indicate that a second conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes. An emerging concept suggests that the 11 universal GTPases are either necessary for ribosome function or transmitting information from the ribosome to downstream targets for the purpose of generating specific cellular responses. Furthermore, it is suggested that progenitor GTPases were early regulators of RNA function and may have existed in precursors of cellular systems driven by catalytic RNA. If this is the case, then a corollary of this hypothesis is that GTPases that do not bind RNA arose at a later time from an RNA‐binding progenitor that lost the capability to bind RNA.

No abstract

No abstract

The DNA sequence of a gene (era) located immediately downstream of the gene (rnc) encoding ribonuclease m of Escherichia coli was determined and found to encode a protein of 316 amino acid residues. The amino acid sequence of this protein, Era, has significant similarity to the yeast RAS proteins. Overexpression of the Era protein was achieved and GTP cross-linking experiments demonstrated that the protein was indeed capable of binding GTP, as are the yeast and mammalian ras gene products. These data indicate that ras-related sequences occur not only in eukaryotes but also in prokaryotes.The ras family of oncogenes was discovered in Harvey and Kirsten murine sarcoma viruses and was subsequently shown to exist in mammalian cells as protooncogenes (for review see refs. 1 and 2). Further investigations demonstrated that certain tumor cells contain structurally mutated ras genes capable of tumorigenic transformation of NIH3T3 cells upon gene transfer (3-7). These findings have led to extensive investigations into the structure and function of ras genes. At least three genes (Ha-, Ki-, and N-ras) have been discovered in mammals and found to encode highly related proteins (8-10). In addition, DNA hybridization experiments have shown that organisms as widely divergent as Drosophila (11,12), Dictyostelium (13), and Saccharomyces (14, 15) possess similar sequences. The same methodology was employed to demonstrate that the fission yeast Schizosaccharomyces contains DNA at a single locus that is highly related to ras (16). Interestingly, Saccharomyces has two genes (RAS] and RAS2), which encode proteins that are homologous to mammalian ras proteins (14,15), and also another gene (YP2) encoding a protein that is less similar (17). Thus far, however, such observations have not been reported in bacteria.We report here that Escherichia coli has a gene (era) that encodes a protein containing significant similarity to the yeast RAS proteins. Since mammalian and yeast ras gene products are known to bind GTP (18,19), GTP cross-linking experiments were performed, and they revealed that the Era protein is a GTP-binding protein as well. DNA Sequencing. The era gene was obtained from the plasmid pSB, which contains a 4-kilobase (kb) EcoRI fragment obtained from the E. coli chromosome. This plasmid has the ability to complement genetic defects in a ribonuclease III-negative background (H.E.T. and D. Court, unpublished data). Fragments for sequencing were obtained by appropriate restriction enzyme digestion (as indicated in Fig. 1) followed by preparative polyacrylamide gel electrophoresis. The DNA fragments were cloned in sequencing vectors M13mp18 or M13mp19 by using the host strain JM103 (available from Bethesda Research Laboratories). DNA sequencing was performed by using the chain-termination method (23) except where indicated in Fig. 1, in which case the method of Maxam and Gilbert (24) was employed to sequence the cloned fragment. MATERIALS AND METHODSExpression of the rnc Locus. The rnc locus was cloned in a high-expres...

Era is a membrane-associated GTP-binding protein which is essential for cell growth in Escherichia coli. In order to examine the physiological role of Era, strains in which Era was expressed at 40°C but completely repressed at 27°C were constructed. The growth of these strains was inhibited at the nonpermissive temperature, and cells became elongated. Under such conditions, no constrictions or septum formation could be detected by phase-contrast microscopy, and DNA segregation was apparently normal as revealed by fluorescence staining. These data demonstrate that Era has an essential function in cell growth rate control in liquid media and that depletion of Era blocks cell division either directly or indirectly. Thus, the role of GTPbinding proteins as important regulators of cell growth and division may be ubiquitous in nature.It is now widely recognized that a large family of similar proteins possessing GTPase activity are key regulators of cell growth and division. These proteins include factors required for protein synthesis, G proteins, and the ras oncogenes, among others. Only a few such GTP-binding proteins are known in Escherichia coli, including a membrane-associated GTP-binding protein (Era) which was shown to possess low-level intrinsic GTPase activity (1,8,16). The gene (era) for this protein has been cloned, sequenced, and located at min 55 on the E. coli chromosome within the rnc operon (1). Mutational analysis has shown that an intact era allele is required for growth on plates, but analysis of conditional mutants has not led to the delineation of any phenotype that suggests a function for Era (11,16,21). In order to understand the function of Era in vivo, we constructed several strains in which transcription of era is controlled by the phage X promoter PR. The strategy employed in mutant construction allows one to obtain a new strain by simply transforming bacteria with the vector harboring the disrupted gene. era mutants incubated at the nonpermissive temperature are nonviable and exhibit an elongated cell phenotype.MATERIALS AND METHODS Strains. The bacterial strains employed to construct conditional era mutants have been described elsewhere and are referred to in Table 1.Growth conditions. Cells were grown in Luria broth media (5 g of yeast extract, 10 g of tryptone [Difco], and 5 g of NaCl per liter). The media were supplemented with antibiotics at the following concentrations in micrograms per milliliter: ampicillin, 50; kanamycin sulfate, 50; and tetracycline, 15.Shift-down experiments were carried out in two water bath shakers with platforms rotating at the same speed. Growth was monitored turbidimetrically by using a KlettSummerson calorimeter containing a red 66 filter.Strategy of mutant construction. The basic strategy involved constructing a temperature-sensitive vector which would permit survival of recipients only when there was a single homologous recombination between the plasmid and * Corresponding author. the targeted gene while at the same time disrupting the target.The plasmid ...