Lifetime of bacterial messenger ribonucleic acid

Moses, V. (University of California, Berkeley), and M. Calvin. Lifetime of bacterial messenger ribonucleic acid. J. Bacteriol. 90:1205-1217. 1965.-When cells from a stationary culture of Escherichia coli were placed in fresh medium containing inducer for beta-galactosidase, growth, as represented by increase in turbidity and by total protein synthesis, started within 30 sec. By contrast, beta-galactosidase synthesis was greatly delayed compared with induction during exponential growth. Two other inducible enzymes (d-serine deaminase and l-tryptophanase) and one repressible enzyme (alkaline phosphatase) showed similar lags. The lags were not due to catabolite repression. They could not be reduced by pretreatment of the culture with inducer, or by supplementing the fresh medium with amino acids or nucleotides. The lag was also demonstrated by an i(-) mutant constitutive for beta-galactosidase synthesis. An inhibitor of ribonucleic acid (RNA) synthesis, 6-azauracil, preferentially inhibited beta-galactosidase synthesis compared with growth in both inducible and constitutive strains. Puromycin, an inhibitor of protein synthesis, acted as an inhibitor at additional sites during the induction of beta-galactosidase synthesis. No inhibition of the reactions proceeding during the first 20 sec of induction was observed, but puromycin seemed to prevent the accumulation of messenger RNA during the period between 20 sec and the first appearance of enzyme activity after 3 min. It is suggested that these observations, together with many reports in the literature that inducible enzyme synthesis is more sensitive than total growth to some inhibitors and adverse growth conditions, can be explained by supposing that messenger RNA for normally inducible enzymes is biologically more labile than that for some normally constitutive proteins. The possible implications of this hypothesis for the achievement of cell differentiation by genetic regulation of enzyme synthesis are briefly discussed.     

Influence of Polyamine Limitation on the Chain Growth Rates of β-Galactosidase and of Its Messenger Ribonucleic Acid

The rates of elongation of beta-galactosidase and its messenger ribonucleic acid (RNA) were estimated in a polyamine-deficient mutant of Escherichia coli through an analysis of the kinetics of enzyme induction. The chain growth of beta-galactosidase was calculated from the time required after the appearance of an amino terminal fragment of 60 amino acids (auto-alpha) until completed enzyme began to accumulate. The elongation rate of beta-galactosidase messenger RNA was estimated from the time after induction at which streptolydigen-resistant, enzyme-forming capacity first appeared. Upon polyamine starvation, the rate of polypeptide elongation slowed from 17 to 10 amino acids per s and the messenger RNA elongation rate decreased from 47 to 30 nucleotides per s. These reductions in polymerization rates were proportional to the decrease in cellular growth rate produced by polyamine starvation. It was concluded that, although it is quite unlikely that polyamine levels are involved in regulation of cell growth, they may be acting as cofactors in the synthesis of RNA or protein, or both.     

Discontinuous synthesis of lac messenger ribonucleic acid in a mutant of Escherichia coli with a new lac promoter.

A mutant of Escherichia coli with a new promoter for the lac operon exhibits dramatic discontinuities in the synthesis of lac messenger ribonucleic acid after induction. These discontinuities immediately precede similar discontinuities in the synthesis of beta-galactosidase. The discontinuous synthesis of beta-galactosidase persists after addition of rifampin.     

Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli

Ben-Hamida, Fakher (Washington University School of Medicine, St. Louis, Mo.), and David Schlessinger. Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli. J. Bacteriol. 90:1611-1616. 1965.-Synthesis of beta-galactosidase stops within several minutes when preinduced, permeaseless cultures are diluted into medium containing 40 mug/ml of 5-fluorouracil (5-FU) but no inducer. However, if inducer (isopropylthiogalactoside) is left in the medium, enzyme formation in the presence of 5-FU continues for at least 11 min. Thus, inducer may increase the differential metabolic stability of the corresponding messenger ribonucleic acid (RNA; defined as the capacity to produce measurable enzyme) in inducible strains. However, such an interpretation requires that 5-FU rapidly arrest the further synthesis of messenger RNA competent to form active enzyme. C(14)-5-FU, like uracil, does appear to enter cells without measurable lag, saturating the pool of uracil nucleotides, and thereby the messenger RNA being formed, within several minutes. That 5-FU acts very quickly is also supported by the similar continuation of enzyme synthesis in the presence of inducer and antibiotics (actinomycin D and proflavine) which shut off all RNA synthesis, as well as by the response to 5-FU of enzyme synthesis in various constitutive mutants.     

Control of protein synthesis in Escherichia coli: strain differences in control of translational initiation after energy source shift-down.

We have studied the parameters of protein synthesis in a number of Escherichia coli strains after a shift-down from glucose-minimal to succinate-minimal medium. One group of strains, including K-12(lambda) (ATCC 10798) and NF162, showed a postshift translational yield of 50 to 65% and a 2- to 2.5-fold increase in the functional lifetime of general messenger ribonucleic acid. There was no change in the lag time for beta-galactosidase induction in these strains after the shift-down. A second group, including W1 and W2, showed no reduction in translational yield, no change in the functional lifetime of messenger ribonucleic acid, and a 50% increase in the lag time for beta-galactosidase induction. Evidence is presented which indicates that this increased lag time is not the result of a decreased rate of polypeptide chain propagation. A third group of strains, including NF161, CP78, and NF859, showed an intermediate pattern: translational yield was reduced to about 75% of normal, and the messenger ribonucleic acid functional lifetime was increased by about 50%. Calculation of the relative postshift rates of translational initiation gave about 0.2, 1.0, and 0.5, respectively, for the three groups. There was no apparent correlation between the ability to control translation and the genotypes of these strains at the relA, relX, or spoT loci. Measurements of the induction lag for beta-galactosidase during short-term glucose starvation or after a down-shift induced by alpha-methylglucoside indicated that these regimens elicit responses that are physiologically distinct from those elicited by a glucose-to-succinate shift-down.     

Transient repression of catabolite-sensitive enzyme synthesis elicited by 2,4-dinitrophenol.

Transient inhibition of catabolic enzyme synthesis in Escherichia coli occurred when a low concentration of 2,4-dinitrophenol (DNP) was simultaneously added with inducer. Using mutant strains defective for gamma-gene product or constitutive for lac enzymes, it was found that the inhibition is not due to the exclusion of inducer by uncoupling. The addition of cyclic adenosine 3',5'-monophosphate overcame repression. The components of the lac operon coordinately responded to DNP inhibition. From deoxyribonucleic acid-ribonucleic acid hybridization experiments, it was found that the inhibition of beta-galactosidase induction occurred at the level of messenger ribonucleic acid synthesis specific for the lac operon. It seems probable that DNP represses induction in a similar manner to that of transient repression observed upon the addition of glucose. Furthermore, it was found that transient repression disappeared if cells were preincubated with DNP before induction. This indicates that new contact of cells with DNP is obligatory for transient repression. From these results, it is suggested that the cell membrane may be responsible for regulation of catabolite-sensitive enzyme synthesis.     

Action of phyenethyl alcohol on the synthesis of macromolecules in Escherichia coli

Prevost, C. (University of California, Berkeley), and V. Moses. Action of phenethyl alcohol on the synthesis of macromolecules in Escherichia coli. J. Bacteriol. 91:1446-1452. 1966.-A kinetic study of the effects of various concentrations of phenethyl alcohol on the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), protein, and beta-galactosidase in Escherichia coli has confirmed that RNA synthesis, rather than DNA synthesis, is first and most affected by phenethyl alcohol. The presence of inducer did not protect beta-galactosidase synthesis from inhibition by phenethyl alcohol. Little preferential inhibition of beta-galactosidase synthesis was observed; this is in contrast to the severe catabolite repression which results from partial inhibition of total protein synthesis caused by chloramphenicol or starvation for a required amino acid. We found no evidence that messenger RNA synthesis was inhibited to a greater extent than total RNA synthesis.     

Bacteriophage-induced Inhibition of Host Functions: II. Evidence for Multiple, Sequential Bacteriophage-induced Deoxyribonucleases Responsible for Degradation of Cellular Deoxyribonucleic Acid

Degradation of bacterial deoxyribonucleic acid (DNA) after infection with T4 bacteriophage was studied in an endonuclease I-deficient host. The kinetics of degradation were similar to those seen in other hosts with a normal level of this enzyme. Irradiation of extracellular phage with ultraviolet (UV) destroyed the capacity of the infecting virus to induce extensive breakdown of host DNA, which was, however, converted to high-molecular-weight material. Addition of chloramphenicol to T4-infected cells provided data which can be interpreted to indicate the involvement of at least two endodeoxyribonucleases and one exodeoxyribonuclease having a high degree of specificity. A model is proposed showing the sequential action of two endodeoxyribonucleases followed by an exodeoxyribonuclease in the degradation of host DNA. The appearance of these hydrolytic enzymes requires protein synthesis. Infections leading to partial degradation only (UV-irradiated phages, gene 46 mutants) effectively inhibited the synthesis of bacterial messenger ribonucleic acid and of beta-galactosidase.     

Inhibition of Host Protein Synthesis During Infection of Escherichi coli by Bacteriophage T4: III. Inhibition by Ghosts

Deoxyribonucleic acid (DNA)-less T2 "ghosts" were prepared by osmotic shock and purified by KBr density gradient centrifugation. Escherichia coli B was treated with these ghosts in inorganic salts-glycerol medium to see which features of phage infection could be elicited by ghosts. At a multiplicity that was just sufficient to block induction of beta-galactosidase (EC 3.2.1.23), 89% of the bacteria were killed and the rates of ribonucleic acid (RNA) and DNA synthesis were about 10 to 15% of normal. However, protein synthesis was almost completely blocked but resumed after 30 min. During this period, it was possible to induce messenger RNA (mRNA) from the lactose operon, although this mRNA could not be translated into active beta-galactosidase. These results suggest to us that the viable cells surviving ghost infection synthesize nucleic acids at close to a normal rate but are temporarily blocked in protein synthesis. The continued formation of untranslated host mRNA mimics the pattern of bacterial synthesis just after whole-phage infection, and is consistent with the interpretation that the immediate block in the initiation of host translation by these viruses is due to their attachment.     

Sequence of molecular events involved in induction of allophanate hydrolase.

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.     

Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.     

Inhibition of lacZ Gene Translation Initiation in trp-lac Fusion Strains

Different levels of beta-galactosidase are found in various trp-lac fusion strains. These levels of beta-galactosidase fall within a 60-fold range. The amount of thiogalactoside transacetylase activity detected in these same strains only varies 10-fold and is found in amounts greater than those predicted from the beta-galactosidase levels. The observation that the beta-galactosidase and thiogalactoside transacetylase levels are not directly proportional, that the lacZ messenger ribonucleic acid (mRNA) levels are not proportional to the beta-galactosidase activity, that, at least for the one fusion strain tested, the SuA polarity suppressor does not affect the beta-galactosidase level, and that, in all but one strain, the beta-galactosidase activity appears to reside in normal beta-galactosidase molecules suggests that the disproportionately low production of beta-galactosidase is due to a decrease in the frequency of translation initiation of lacZ mRNA in these strains. Several mechanisms are proposed to explain this decrease. Some possible bases for the disproportional production of beta-galactosidase and thiogalactoside transacetylase are also described. The preferred explanation for these disproportional enzyme levels is that only a fraction of the full complement of ribosomes need initiate translation at lacZ for the functional synthesis of lac mRNA to occur and that once the lac ribonucleic acid is made a full complement of ribosomes can bind at internal translation initiation sites at Y and A.     

Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase.

Yeast poly(adenylic acid)-containing messenger ribonucleic acid isolated from two strains of Saccharomyces cerevisiae was fractionated by preparative polyacrylamide gel electrophoresis in the presence of formamide. Three messenger ribonucleic acids, present at high intracellular concentration, were electrophoretically eluted from the polyacrylamide gels and translated in a wheat germ cell-free extract. The in vitro synthesized polypeptides were identified by tryptic peptide analysis. Messenger ribonucleic acids coding for enolase and glyceraldehyde-3-phosphate dehydrogenase were isolated from commercially grown baker's yeast (strain F1), and messenger ribonucleic acid coding for phosphoglycerate kinase was isolated from Saccharomyces cerevisiae (ATCC 24657). Significant differences in the spectrum of abundant messenger ribonucleic acids isolated from commercially grown baker's yeast (strain F1) and strain 24657 were observed. When both strains were grown under identical conditions, however, the spectrum of messenger ribonucleic acid isolated from the cells is indistinguishable.     

Kinetics of the onset of catabolite repression in Escherichia coli as determined by lac messenger ribonucleic acid initiations and intracellular cyclic adenosine 3'',5''-monophosphate levels.

The rates of synthesis of beta-galactosidase (EC 3.2.1.23) and the intracellular levels of cyclic 3',5'-adenosine monophosphate (cAMP) soon after the addition of glucose or glycerol to exponentially growing cultures of Escherichia coli have been determined. Within 10 s of its addition, glucose, but not glycerol, lowered the apparent initiation frequency of lac messenger ribonucleic acid. The glucose-generated reduction in initiations is identified as catabolite repression by its reversibility with cAMP. The intracellular cAMP levels respond virtually identically to glucose and glycerol additions. Thus, no correlation was observed between the rate of messenger ribonucleic acid initiation and the level of cAMP.