Regulation of mRNA Utilization and Degradation by Amino-acid Starvation

Amino-acid starvation of a stringent strain of E. coli relieves the polar effect on distal messenger RNA imposed by a nonsense mutation. There is no relief of polarity starvation of a relaxed strain. In fact, starvation of a relaxed (but not of a stringent) strain by itself causes an artificial polar effect on distal mRNA. These findings are consistent with a mechanism for polarity based on mRNA degradation.     

Functional aspects of bacterial polysomes during limited protein synthesis

The effects of amino acid starvation on the metabolic behavior of polysomes and the size distribution of proteins have been studied in an otherwise isogenic pair of stringent (relA+) and relaxed (relA) strains of Escherichia coli. The stability of polysomes has been analyzed by using two different approaches. First, the process of their degradation has been followed after treating the cells with rifampicin, an inhibitor of the synthesis of all classes of RNA including messenger RNA. Secondly, the process of their assembly has been studied after their previous conversion to monosomes, as induced by glucose deprivation of cells. It is shown that, in either type of bacterial strain, polysomes are continually broken down and re-synthesized during amino acid starvation. However, such polysome turnover is then less rapid than in normally growing bacteria and, moreover, it seems amino acid specific since it occurs at a lower rate during arginine starvation than during histidine starvation, namely, in the relaxed strain. The molecular weight distribution of proteins has been determined after labeling of cells with radioactive methionine and separation of polypeptides by one-dimensional polyacrylamide gel electrophoresis. The average size of polypeptides synthesized in the stringent strain during starvation is quite similar to that measured during normal growth. By contrast, a significant shift towards smaller molecules is observed in the relaxed strain deprived of an essential amino acid. Here again, this reduction of the size of polypeptides seems amino acid specific since it is especially marked during arginine starvation. These results are discussed in terms of ribosomes translocation and premature peptide chain termination in connection with the accuracy of the translational process.     

Synthesis, no synthesis, or synthesis and degradation of ribosomal ribonucleic acid in various Escherichia coli strains starved for an amino acid

A standard stringent strain of Escherichia coli makes little or no ribosomal ribonucleic acid (RNA) during starvation for an essential amino acid, whereas the isogenic relaxed strain makes both ribosomal and messenger RNA. A third class of strains was found which continues to make ribosomal RNA during starvation, but the RNA made is apparently unstable. There is little accumulation of RNA in the third class of strains, and few complete newly formed chains of (3)H-ribosomal RNA are observed in sedimentation analyses, even after long labeling times.     

Relaxed mutants ofSerratia marcescens SM-6. Biochemical traits and relevance of therel + allele for the formation of exoenzymes

Serratia marcescens SM-6 when starved for a required amino acid stops synthesizing protein and RNA and accumulates two nucleotides which cochromatograph with ppGpp and pppGpp. These features are characteristic of bacterial strains with stringent RNA control (rel ⁺). Two independent mutants were isolated which resemble relaxed (relA) mutants ofEscherichia coli; they continue to synthesize RNA and accumulate neither ppGpp nor pppGpp when deprived of the required amino acid. The extracellular enzyme activities (nuclease, protease, lipase) of the relaxed mutants are about the same as those of the parental stringent strain when studied under standard growth conditions. Exoenzyme-deficient (nuc; prt) and exoenzyme-hyperproducing (nuc ^su) mutants were isolated from both stringent and relaxed strains ofS. marcences SM-6 and no change of the cellular ability to form ppGpp and pppGpp could be observed. From these results it appears that the formation of exoenzymes ofS. marcescens SM-6 is independent of stringent/relaxed RNA control.Abbreviations cpd cyclic nucleotide phosphodiesterase deficient - nuc nuclease deficient - nuc ^su nuclease hyperproducing - prt protease deficient - rel relaxed control - spo ppGpp deficient (spot less) - ppGpp guanosine tetraphosphate - pppGpp guanosine pentaphosphate - TCA trichloroacetic acid - OD optical density - EU enzyme units     

Movement of ribosomes over messenger RNA in polysomes of rel + and rel - Escherichia coli strains

The in vitro movement of ribosomes over messenger RNA was studied in both the presence and the absence of protein synthesis. For this purpose, labeled polysomes were extracted from rel⁺ and rel^? strains of Escherichia coli grown in the presence of radioactive uracil and incubated in a cell-free system containing tRNA, amino acids, soluble enzymes and a source of energy. The gradual conversion of the labeled polysomes into monosomes and ribosomal subunits was followed by subjecting the reaction mixture to sucrose gradient sedimentation after various incubation times and measuring the radioactivity present in the three relevant ribosomal fractions.It was found that when the conditions of incubation allow protein synthesis to occur, polysomes extracted from rel⁺ and rel^? cells are converted mainly into free monosomes, which can be made to dissociate into subunits by high-sodium or low-magnesium ion concentrations. Under conditions in which protein synthesis cannot occur because a mutant aminoacyl-tRNA synthetase has been rendered inactive, polysome conversion still occurs, though to a reduced extent. When the products of such residual run-off are examined, however, a difference is manifest between polysomes extracted from rel⁺ and from rel^? strains: whereas the polysomes from the rel^? strain are still converted into free monosomes even in the absence of protein synthesis, the polysomes from the rel⁺ strain are now converted mainly into subunits. It can be inferred, therefore, that ribosomes from rel^? bacteria, but not those from rel⁺ bacteria, continue movement over messenger RNA in the absence of protein synthesis.Studies of mixed extracts from rel^? and rel⁺ bacteria have shown that the character of the run-off process does not depend on the source of tRNA and soluble enzymes; the proportions of monosomes and subunits among the run-off products formed in the absence of protein synthesis depend only on the source of the polysomes. It is suggested that the mutation of the rel gene alters the functional architecture of ribosomes.     

Isolation and characterization of prototrophic relaxed mutants of Klebsiella pneumoniae

Summary A new selection procedure has been developed for isolating prototrophic relaxed mutants of Klebsiella pneumoniae. Two mutants were isolated. One of them showed a fully relaxed phenotype, while the other one behaved in a semi-relaxed way.The wild-type strain, as well as the rel mutants exerted similar patterns to their E. coli counterparts in RNA, protein, ppGpp and pppGpp accumulation during amino starvation, carbon source shift-down and nitrogen starvation. Both mutants became stringent after introducing an F-factor carrying the relA ⁺ allele from Escherichia coli. The relaxed phenotype could be recovered by curing the F-factor. Some of the pleiotropic consequences of rel mutations found in E. coli are present in the Klebsiella mutants also while some of them are absent.The mutants are defective in dinitrogen fixation after the exhaustion of limiting ammonium from the culture medium. However, their merodiploid derivatives, carrying the E. coli relA ^- allele, showed the wild-type level of nitrogenase activity under the same conditions.Fellow of the 6th International Training Course jointly sponsored by UNDP/UNESCO Hungarian Academy of Sciences. Present address: Akademie der Wissenschaften der DDR, Forschungszentrum für Molekularbiologie und Medizin, Zentralinstitut für Mikrobiologie und Experimentelle Therapie Jena, Beuthenberg Str. 11, DDR-69 Jena     

Regulation of stable RNA synthesis and ppGpp levels in growing cells of Escherichia coli

Under the balanced condition of growth of E. coli cells, no distinct difference is observed in stable RNA and protein synthesis between CP78 (rel⁺) and CP79 (rel⁻), whereas a considerable difference is present in RNA accumulation between NF161 (rel⁺) and NF162 (rel⁻), where NF161 < NF162. The RNA content of NF161 is lower than that of NF162 in four different cultures with different growth rates. These two sets of isogenic pairs of rel⁺ and rel⁻ strains are commonly used in the study of rel gene function; however, NF161 is a mutant in the spoT gene whose product may be responsible for the degradation of ppGpp. The basal levels of ppGpp in these four strains growing with three different growth rates were examined: NF161 (rel⁺spoT⁻) has a much higher content of ppGpp than do other strains. Furthermore, the contents of ppGpp tend to be lower when the above four strains are growing at a faster rate. Thus a close correlation seems to exist between the content of RNA and the basal level of ppGpp under the condition of balanced growth.     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.     

Thermolability of the stringent factor in rel mutants of Escherichia coli

The stringent factor extracted from several independently isolated rel^- mutants is more thermolabile than the stringent factor extracted from the parental rel⁺ strain. This thermolability is characteristically different in each of the mutants. This strongly suggests that the stringent factor is the product of the rel gene.     

Cyclic AMP can replace the relA-dependent requirement for derepression of acetohydroxy acid synthase in E. coli K-12.

Derepression of the isoleucine and valine biosynthetic enzymes was strongly impaired in a relA strain of E. coli K-12 grown in an amino acid-glucose medium. The expression of the isoleucine and valine operons during leucine starvation was markedly defective in the relA mutant as compared to an isogenic rel⁺ strain. Downshift to a poor carbon and energy source or the addition of cyclic AMP to the glucose medium allowed normal derepression in the relA mutant of one of the isoleucine and valine enzymes, acetohydroxy acid synthase. The other isoleucine and valine enzymes failed to derepress under these conditions, in contrast to the high enzyme levels in the rel⁺ parent. No increase in acetohydroxy acid synthase was found in relA cya or relA crp strains during glycerol or pyruvate downshift. Cyclic AMP allowed derepression in the relA cya mutant but not in the relA crp strain. These data strongly suggest that the relA requirement for normal expression of acetohydroxy acid synthase can be replaced by cyclic AMP.     

Cell volume change of Escherichia coli under stringent control apparent increase of K in rel cells

With several pairs of rel⁺ and rel⁻ strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K⁺ and the rate of uptake of ⁴²K⁺ were investigated. In the early phase of the experiments, the intracellular concentration of K⁺ was estimated by the conventional method in which the cell volume per A₆₆₀ value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K⁺ concentration of rel⁺ cells was kept almost constant, while that of rel⁻ cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A₆₆₀ changed only slightly in CP78 (rel⁺) cells, while it increased markedly in CP79 (rel⁻) cells after the exposure to amino acid starvation. Reestimation of the K⁺ concentrations based on the estimated respective values of cell volumes per A₆₆₀ revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel⁺ cells was arrested upon amino acid starvation whereas that in the rel⁻ cells was not. The ⁴²K⁺ uptake by the rel⁺ cells was depressed upon amino acid starvation, whereas that by the rel⁻ cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K⁺ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A₆₆₀ of the culture, was pointed out.     

Use of the Mud(Ap,lac) bacteriophage to study the regulation of L-proline biosynthetic genes inEscherichia coli K-12

One operon fusion to the promoter of either theproA orproB genes of the proline biosynthetic pathway was obtained by the use of the Mud(Ap,lac) bacteriophage. This operon fusion was further stabilized by transformation with the plasmid pGW600 containing the wild type Mu repressor gene. The level of β-galactosidase in this strain was not affected by the presence of high concentrations of NaCl in the growth medium. Mutations affecting the regulation of thispro-lac genetic fusion were generated by the insertion of Tn5; β-galactosidase levels in these mutants were higher than in the parental strain when proline was present at a high level. In some of these mutants we observed either repression or maintenance of β-galactosidase levels whenpro-lac (F′proAB ⁺) merodiploids were constructed.     

Isolation of relaxed-control mutants of Escherichia coli K-12 which are sensitive to glucose starvation

Mutants of $\text{Escherichia coli}$ K-12 which are sensitive to glucose starvation were isolated by an enrichment procedure using thymine starvation to select for nongrowing cells. Eleven independent isolates were obtained by this method. The mutants are also sensitive to glycerol starvation and to a lesser extent to nitrogen or amino acid starvation. The mutants are more sensitive than the parental strain to inhibitors of protein synthesis but not inhibitors of RNA or DNA synthesis. [³H]-leucine incorporation experiments indicate that protein synthesis is blocked in the mutants during recovery from glucose starvation or chloramphenicol inhibition. Incorporation of [³H]uridine in amino acid-starved cells demonstrates that the mutants are partially relaxed for control of RNA synthesis. Physiological and genetic experiments indicate that these mutants are different from previously isolated relaxed-control mutants.     

A conditional lethal mutation in an Escherichia coli strain with a longer chemical lifetime of messenger RNA.

We have analysed an Escherichia coli temperature-sensitive mutant with altered messenger RNA stability, and it was found that: (1) the unstable fraction of pulse-labelled RNAs decayed with a half-life at 42 °C of about two minutes in the parent strain PA3092; the half-life was 11 to 12 minutes in the mutant HAK75. Puromycin enhanced the decay rate about twofold in both PA3092 and HAK75; the addition of chloramphenicol inhibited the degradation significantly in both strains. The rate of ribosomal RNA accumulation in the mutant cells at 42 °C did not differ from that in the wild-type cells. (2) Sedimentation analysis by sodium dodecyl sulphate/sucrose density-gradient centrifugation of bulk mRNA as well as tryptophan mRNA of the wild-type strain showed the expected rapid reduction in the size and level of those mRNA molecules at three minutes and five minutes respectively, after addition of rifampicin at 42 °C. In contrast, the cells of HAK75 retained almost full-length trp mRNA and bulk mRNA at 5 to 12 minutes after the addition of rifampicin at 42 °C, even though the total level of radioactivity in the mRNA fraction had decreased to about 60 to 75% of the initial activity. (3) Even though mRNA molecules were chemically protected at the non-permissive temperature in the mutant, the functional decay of both β-galactosidase and tryptophan synthetase occurred at a rate comparable to that in the parental strain. (4) We isolated temperature-resistant revertants from the mutant at a frequency of 5 × 10^?8, and these revertants (TR1 and TR2) had the normal decay rate of unstable RNA.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.