Role of guanosine tetraphosphate in gene expression and the survival of glucose or seryl-tRNA starved cells of Escherichia coli K12

The concentration of guanosine 3,5-bispyrophosphate (ppGpp) increases in bacteria in response to amino acid or carbon/energy source starvation. An Escherichia coli K12 relAspoT mutant lacking the ability to synthesize ppGpp lost viability at an increased rate during both glucose and seryl-tRNA starvation. Also, the deleterious effect of chloramphenicol on starved wild-type cells could be overcome by inducing expression of RelA from a plasmid carrying the relA gene transcribed from a tac promoter, prior to starvation and chloramphenicol treatment. As demonstrated by two dimensional gel electrophoresis, this induction of the RelA protein resulted in global alterations in gene expression including increased synthesis of some rpoS-dependent proteins. The relAspoT mutant maintained high expression of several ribosomal proteins during starvation and appeared to exhibit significantly decreased translational fidelity, as demonstrated by an unusual heterogeneity in the isoelectric point of several proteins and the failure to express higher molecular weight proteins during starvation. Moreover, both rpoS-dependent and independent genes failed to exhibit increased expression in the mutant. It is suggested that the deleterious effects on the cells of the relA, spoT deletions are not due solely to the inability of these cells to induce the sigma factor ^s, but also to deficiencies in translational fidelity and failure to exert classical stringent regulation.     

Outwitting EF-Tu and the ribosome: translation with d-amino acids

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNA^Gly that was selected according to the rules of ‘thermodynamic compensation’. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.     

Positive involvemetn of ppGpp in derepression of the nif operon in Klebsiella pneumoniae

Summary The kinetics of derepression of the enzyme nitrogenase were investigated, after exhaustion of a limiting amount of ammonium from the culture medium, in a prototrophic stringent-relaxed pair of Klebsiella pneumoniae strains and in their F relA ⁺-F relA derivatives. The results indicate that ppGpp (guanosine 3–5 diphosphate) increases the nitrogen fixation capability of K. pneumoniae by at least three different mechanisms. (1) It prevents exhaustion of the ATP pool when nitrogen starvation is imposed. (2) The translational defects in relaxed mutants are suppressed by ppGpp during nif derepression. (3) The synthesis of nitrogenase components is at least five times higher in the presence of ppGpp than in its absence. This latter conclusion was based on experimental results obtained when following the incorporation of (³⁵S)-methionine into nitrogenase components after pulse labelling at various time intervals during nif derepression. The nitrogenase components were separated by solid phase radioimmunoassay as well as by two-dimensional gel electrophoresis.     

Autogenous regulation of the synthesis of ribosomal proteins, L10 and L7/12, in Escherichia coli

Summary The in vitro synthesis of Escherichia coli ribosomal proteins, L10 and L7/12, is specifically repressed by the addition of the L10-L7/12 complex, while that of other ribosomal proteins encoded by the neighboring operons is not affected. Thus the expression of the rpoBC operon is controlled by two autorepression systems, one for the two ribosomal proteins and the other for RNA polymerase and subunits, both operating probably at the translational level.     

Physical mapping of mutations in the structural gene encoding for the Escherichia coli aldohexuronate transport system

Summary Synthesis of both chromosomal and plasmid (pBR322) DNA was measured in E. coli strains differing in their relA allele (relA ⁺: CP78; relA: CP79). It was found that upon limitation of a required amino acid or after valine treatment to trigger a stringent response synthesis of pBR322 DNA was stimulated only in the relaxed strain and was inhibited in its stringent counterpart. The results suggest that replication of plasmid DNA is negatively controlled by the relA ⁺ allele.     

Decay and synthesis of ribosomal proteins during Dictyostelium discoideum development

Summary Ribosome turnover is a prominent process during cell differentiation in Dictyostelium discoideum. At the end of 24 h of development on filters, the cells contain only 30% of the ribosome content of vegetatively growing cells. We determined the relative rates of synthesis and decay of each of the ribosomal proteins during this period. Approximately 80% of the total vegetative cell ribosomal proteins were degraded during the course of fruiting body construction. Ribosomal RNA and protein degradation apparently occurred coordinately during development. Although all ribosomal proteins decayed during development, some were more stable and a few less stable than the average. In addition, all the ribosomal proteins were synthesized during this period. Most ribosomal proteins were synthesized at the same rate as other cellular proteins, although a number were made at lower or higher rates. It was estimated that about 35% of the ribosomes in developed cells represented those, that were made during cell differentiation. Differential decay and/or synthesis of ribosomal proteins could account for the observed difference in protein content of ribosomes from growing amoebae and late development cells and spores.Paper No. 4 in the series, Studies on Ribosomal Proteins in Dictyostelium discoideum. Paper No. 3 is Ramagopal and Ennis (1982)     

Influence of stringent and relaxed response on excretion of recombinant proteins and fatty acid composition in Escherichia coli

In contrast to stringent (relA⁺) cells of Escherichia coli, relaxed (relA) cells excreted recombinant proteins (-lactamase, interferon 1) into the culture medium during amino acid limitation. Comparative analyses of overall fatty acid composition in relA⁺ cells and relA cells were performed and revealed that, in wild-type cells, drastic alterations occurred during the stringent response. The portion of saturated fatty acids (C16:0) and the fractions of cyclopropane fatty acids (C17_cyc and C19_cyc) increased whereas the portions of unsaturated fatty acids (C16:1 and C18:1) decreased. In cells of the relaxed mutant, no significant changes in the overall composition of the fatty acids were observed after the onset amino acid limitation. These results indicate that a change in fatty acid composition of membrane lipids after starvation of cells may be responsible for the prevention of loss of cellular proteins into the culture medium in stringent controlled cells of Escherichia coli.     

Unique antibiotic sensitivity of an in vitro polypeptide synthesis system from the archaebacterium Thermoplasma acidophilum. Phylogenetic implications

Summary The susceptibility of Thermoplasma acidophilum (an extremely acidophilic, moderately thermophilic, wall-less sulphur-oxidizing archaebacterium) to 50 ribosome-specific inhibitors of polypeptide elongation was surveyed using efficient poly(U)-and poly(UG)-directed cell-free systems and comparable reference systems derived from eubacterial (Bacillus stearothermophilus, Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) species. Under optimum temperature (58° C) and ionic conditions for polypeptide synthesis Thermoplasma ribosomes are only sensitive to the 70 S/80 S ribosome-directed aminoglycoside neomycin, and to five 80 S ribosome-directed inhibitors all of which (-sarcin, mitogillin, restrictocin, dianthin and gelonin) impair the functioning of the large (60 S) ribosomal subunit. Sensitivity of the three structurally related compounds -sarcin, mitogillin and restrictocin and susceptibility to neomycin place Thermoplasma ribosomes between those of Sulfolobus solfataricus (only sensitive to -sarcin) and Methanococcus vannielli (sensitive to -sarcin, mitogillin, restrictocin and neomycin but also affected by a variety of 70 S ribosome-directed drugs). The phylogenetic significance of the greatly diversified antibiotic sensitivity spectra displayed by archaebacteria in general, as opposed to the uniform ones exhibited by eubacteria and eukaryotes, is discussed.     

The twenty aminoacyl-tRNA synthetases from Escherichia coli. General separation procedure, and comparison of the influence of pH and divalent cations on their catalytic activities

A general separation procedure of the twenty E. coli aminoacyl-tRNA synthetases including either a 105 000 g centrifugation or a polyethyleneglycol-dextran two-phases partition fractionation, and chromatographies on DEAE-cellulose, phosphocellulose and hydroxyapatite is described. The specific activities of the synthetases have been determined after each chromatographic step and compared to their respective activities in the 105 000 g supernatant. Some aminoacyl-tRNA synthetases were obtained at 80 per cent purity.The presence of phenylmethylsulfonyl fluoride does not significantly modify either the elution patterns of the synthetases during the various chromatographic steps or their specific activities. Thus, contrarily to enzymes from various eukaryotic organisms no significant inactivation of the E. coli aminoacyl-tRNA synthetases occurs via proteolytic processes during the purification procedure.The effects of various factors: pH, magnesium, and other bivalent cations including spermidine, were tested on the aminoacylation and the [³²P] PP_i-ATP isotope-exchange reactions, and the optimal aminoacylation and isotope-exchange conditions determined for 18 of the 20 E. coli aminoacyl-tRNA synthetases.     

Divergence of glutamate and glutamine aminoacylation pathways: Providing the evolutionary rationale for mischarging

Aminoacyl-tRNA for protein synthesis is produced through the action of a family of enzymes called aminoacyl-tRNA synthetases. A general rule is that there is one aminoacyl-tRNA synthetase for each of the standard 20 amino acids found in all cells. This is not universal, however, as a majority of prokaryotic organisms and eukaryotic organelles lack the enzyme glutaminyl-tRNA synthetase, which is responsible for forming Gln-tRNA^Gln in eukaryotes and in Gram-negative eubacteria. Instead, in organisms lacking glutaminyl-tRNA synthetase, Gln-tRNA^Gln is provided by misacylation of tRNA^Gln with glutamate by glutamyl-tRNA synthetase, followed by the conversion of tRNA-bound glutamate to glutamine by the enzyme Glu-tRNA^Gln amidotransferase. The fact that two different pathways exist for charging glutamine tRNA indicates that ancestral prokaryotic and eukaryotic organisms evolved different cellular mechanisms for incorporating glutamine into proteins. Here, we explore the basis for diverging pathways for aminoacylation of glutamine tRNA. We propose that stable retention of glutaminyl-tRNA synthetase in prokaryotic organisms following a horizontal gene transfer event from eukaryotic organisms (Lamour et al. 1994) was dependent on the evolving pool of glutamate and glutamine tRNAs in the organisms that acquired glutaminyl-tRNA synthetase by this mechanism. This model also addresses several unusual aspects of aminoacylation by glutamyl- and glutaminyl-tRNA synthetases that have been observed.Based on a presentation made at a workshop—Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: D. Söll     

DNA-dependent in vitro synthesis of ribosomal proteins,protein elongation factors,and RNA polymerase subunit α: Inhibition by ppGpp

We have previously shown that the synthesis of ribosomal proteins (r proteins) in E. coli cells is under stringent control (Dennis and Nomura, 1974). Since guanosine tetraphosphate (ppGpp) had been implicated in stringent control, we examied the effects of ppGpp on the in vitro synthesis of r proteins directed by DNA from transducing phage λfus3 and λrif^d18. λfus3 carries genes for protein elongation factors EF-Tu and EF-G, and RNA polymerase subunit α, in addition to genes for approximately 27 r proteins. λrif^d18 carries genes for EF-Tu, RNA polymerase subunits β and β^I, and a set of rRNAs, in addition to genes for approximately five r proteins. We have shown that low concentrations of ppGpp (0.2–0.3 mM) specifically inhibit DNA-dependent r protein synthesis in this system, and that this inhibition takes place directly, rather than as a consequence of the inhibition of rRNA synthesis by ppGpp. In addition, we have also shown that ppGpp inhibits the synthesis of EF-G, EF-Tu, and RNA polymerase subunit α, as well as rRNAs.     

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA ^–] bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom ^– in both relA ^– and relA ⁺ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom ^–, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.     

Accumulation of guanosine tetraphosphate induced by polymixin and gramicidin in Escherichia coli

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA⁺) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

Translation elongation factor 3: a fungus-specific translation factor?

Fungi appear to be unique in their requirement for a third soluble translation elongation factor. This factor, designated elongation factor 3 (EF-3), was first described in the yeast Saccharomycescerevisiae and has subsequently been identified in a wide range of fungal species Including Candida albicans and Schizo-saccharomyces pombe. EF-3 exhibits ribosome-dependent ATPase and GTPase activities that are not intrinsic to the fungal ribosome, but which are essential for translation elongation. Recent studies on the structure of EF-3 from several fungal species have shown that it consists of a repeated domain, with each domain containing the expected putative ATP- and GTP-binding motifs. Overall, EF-3 shows striking amino acid similarity to members of the ATP-binding Cassette (ABC) family of membrane-associated transport proteins although EF-3 is not itself directly membrane-associated. Regions of the EF-3 polypeptide also show structural homology with other translation-associated factors including aminoacyl-tRNA synthetases and the Escherichia coli ribosomal protein S5. While the precise role of EF-3 in the translation elongation cycle remains to be defined, recent evidence suggests that it may be involved in optimizing accuracy during mRNA decoding at the ribosomal A site. Furthermore, the essential nature of EF-3 with respect to the fungal cell indicates that it may be an effective antifungal target. Its apparently ubiquitous occurrence throughout the fungal kingdom also suggests that it may be a useful fungal taxonomic marker.     

Isolation and characterization of 5S RNA-protein complexes from Bacillus stearothermophilus and Escherichia coli ribosomes

Summary A native B. stearothermophilus 5S RNA-protein complex was isolated. Homologous and hybrid 5S RNA-protein complexes could be reconstituted from B. stearothermophilus and E. coli 5S RNA and ribosomal proteins. The major proteins involved in these complexes are for the B. stearothermophilus system B-L5 and B-L22 and for the E. coli system E-L18 and E-L25. Furthermore, a two-dimensional electrophoresis pattern of B. stearothermophilus 50S proteins is presented.Paper No. 2 on Structure and Function of 5S RNA. Preceding paper is by Erdmann, V. A., Doberer, H. G., Sprinzl, M., Molec. gen. Genet. 114, 89–94 (1971).     

Structure,function and evolution of seryl-tRNA synthetases: Implications for the evolution of aminoacyl-tRNA synthetases and the genetic code

Two aspects of the evolution of aminoacyl-tRNA synthetases are discussed. Firstly, using recent crystal structure information on seryl-tRNA synthetase and its substrate complexes, the coevolution of the mode of recognition between seryl-tRNA synthetase and tRNA^ser in different organisms is reviewed. Secondly, using sequence alignments and phylogenetic trees, the early evolution of class 2 Amnoacyl-tRNA synthetases is traced. Arguments are presented to suggest that synthetases are not the oldest of protein enzymes, but survived as RNA enzymes during the early period of the evolution of protein catalysts. In this view, the relatedness of the current synthetases, as evidenced by the division into two classes with their associated subclasses, reflects the replacement of RNA synthetases by protein synthetases. This process would have been triggered by the acquisition of tRNA 3 end charging activity by early proteins capable of activating small molecules (e.g., amino acids) with ATP. If these arguments are correct, the genetic code was essentially frozen before the protein synthetases that we know today came into existence. Correspondence to: S. CusackBased on a presentation made at a workshop-Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code-held at Berkeley, CA, July 17–20, 1994