Control of Protein Synthesis in Escherichia coli: Analysis of an Energy Source Shift-Down

The energy source shift-down described in the preceding paper (Molin et al., J. Bacteriol. 131: 7-17, 1977) was used to study the effects of shift-down on protein synthesis. The overall rate of protein synthesis was reduced immediately, and to the same extent, in stringent and relaxed strains. The primary effect of the shift was a slowing down of the polypeptide chain growth rate, a finding not previously reported. In stringent strains the normal, preshift rate was reestablished within 2 to 3 min, whereas in relaxed strains the chain growth rate remained low for about 20 min before slowly returning to the normal value, which was reestablished some 50 to 60 min after the shift. Throughout this transition, the stability of messenger ribonucleic acid (mRNA) remained unchanged in both strains. We interpret these findings as evidence of the more rapid reduction of the mRNA pool in the stringent strain after shift-down: we believe that very soon after the shift, the stringent strain reduces its pool of mRNA and with it the number of ribosomes engaged in protein synthesis. In this manner the number of active ribosomes is adjusted to the availability of energy and carbon. The relaxed strain cannot rapidly reduce its mRNA pool, which thus remains large enough to engage a near-preshift number of ribosomes during a prolonged period; as a consequence its ribosomes must work at a reduced rate. The possibility that ppGpp is involved in the control of mRNA production is discussed. After shift-down, the initial part of beta-galactosidase (the auto-alpha fragment) was produced at a higher rate than complete beta-galactosidase in the relaxed strain, as expected when translation is impeded.     

In vivo regulation of chromosomal beta-lactamase in Escherichia coli.

Chromosomal beta-lactamase, a periplasmic enzyme of Escherichia coli, was studied with respect to its regulation in vivo. Both the activity and the amount of beta-lactamase increased with growth rate. During a nutritional shift-down, chromosomal beta-lactamase activity followed stable ribonucleic acid accumulation. After a nutritional shift-up the differential rate of beta-lactamase synthesis did not increase immediately (like stable ribonucleic acid), but did increase after a lag period of 30 min. To determine whether beta-lactamase was under stringent control, strains carrying a temperature-sensitive valyl-transfer ribonucleic acid synthetase and differing only in the allelic state of the relA gene were shifted from a permissive to a semipermissive temperature. No influence by the relA gene product was found on beta-lactamase synthesis. The regulation of this periplasmic enzyme is discussed in relation to that of some components of the translational apparatus.     

Regulation of Ribonucleic Acid Synthesis in Escherichia coli During Diauxie Lag: Accumulation of Heterogeneous Ribonucleic Acid

The synthesis of ribonucleic acid (RNA) and of protein in Escherichia coli during glucose-lactose diauxie lag have been examined. The rate of RNA synthesis is about 7%, of the corresponding rate during exponential growth and the rate of protein synthesis 10 to 15%. Inhibition of RNA synthesis occurs to the same extent in both rel and rel(+) strains. The RNA which accumulates during 20 min in diauxie lag is composed of about 50% ribosomal and transfer RNA species and about 50% of a fraction which resembles messenger RNA (mRNA) in its heterogeneous sedimentation properties. Decay of the heterogeneous fraction occurs in the presence of glucose and actinomycin D with a half-life of 3 min, the same as that of pulse-labeled mRNA; however, during the diauxie lag, the half-life of this RNA is about 25 min. Accumulation of the heterogeneous RNA is further increased when protein synthesis is blocked by chloramphenicol. The data suggest that the disproportionate accumulation of mRNA during diauxie lag and energy source shift-down may be attributed at least in part to increased stability of mRNA, but do not rule out a preferential synthesis of mRNA.     

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.     

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.     

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.     

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.     

Inhibitor-Induced Shift-Downs in Escherichia coli

A shift-down response in Escherichia coli cells has been brought about by moderate concentrations of azide or cyanide. Early events of the response included a preferential inhibition of ribonucleic acid relative to deoxyribonucleic acid synthesis, a degradation of polyribosomes, and an inhibition of protein synthesis followed by a transient relief. These changes were entirely comparable to those observed with nutrient-induced shift-downs. The influences of various nutrient supplements on an azide shift-down were examined, and methionine was found to relieve effectively the inhibition of ribonucleic acid synthesis in some strains of cells.     

The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli

The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site.     

Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down.

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.     

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.     

Evidence for messenger ribonucleic acid of an ammonium-inducible glutamate dehydrogenase and synthesis, covalent modification, and degradation of enzyme subunits in uninduced Chlorella sorokiniana cells.

The cells of Chlorella sorokiniana cultured in nitrate medium contain no detectable catalytic activity of an ammonium-inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH). However, several lines of experimental evidence indicated that the NADP-GDH messenger ribonucleic acid was present at high levels and was being translated in uninduced cells. First, binding studies with 125I-labeled anti-NADP-GDH immunoglobulin G and total polysomes isolated from uninduced and induced cells showed that NADP-GDH subunits were being synthesized on polysomes from both types of cells. Second, when polyadenylic acid-containing ribonucleic acid was extracted from polysomes from uninduced and induced cells and placed into a messenger ribonucleic acid-dependent in vitro translation system, NADP-GDH subunits were synthesized from the ribonucleic acid from both sources. Third, when ammonia was added to uninduced cells, NADP-GDH antigen accumulated without an apparent induction lag. Fourth, by use of a specific immunoprecipitation procedure coupled to pulse-chase studies with [35S]sulfate, it was shown that the NADP-GDH subunits are rapidly synthesized, covalently modified, and then degraded in uninduced cells.     

Control of protein synthesis in Escherichia coli: Lack of correlation with changes in intracellular pools of ATP,GTP, and ppGpp

Intracellular pools of ATP, GTP, and ppGpp have been measured in Escherichia coli after an energy source shift-down from glucose minimal to succinate-minimal medium. In a Tic⁺ strain (ATCC 10798), which reduces translational initiation after the down-shift, the rate of protein labeling falls to about 30% of its preshift rate within the first minute after shift and reaches a minimum of 17% by 6 min after shift. The ATP pool in this strain remains constant for about 10 min after shift, then declines gradually to about 60% of its initial level. The temporal discrepancy between protein synthesis and the decline in the ATP pool indicates that a decrease in intracellular ATP is not necessary for the control of protein synthesis. In a Tic^? strain (W1), which cannot control translational initiation under these conditions, the decline in the ATP pool is somewhat more rapid and more pronounced (to 40%) than in the Tic⁺ strain, indicating that the decline in the ATP pool is not sufficient to trigger control of translational initiation. The intracellular GTP pool in the Tic⁺ strain remains constant for 2 min after shift, then declines gradually to reach a minimum of 45% of its initial level at 20 min after shift. The pattern is in general similar in the the Tic^? strain, although the ultimate decline in GTP is more pronounced (to 29%). These data indicate that the decline in GTP is not sufficient and probably unnecessary to elicit control of translational initiation. Intracellular levels of ppGpp increase with very similar kinetics in relA⁺Tic⁺ (ATCC 10798) and relA⁺Tic^? (W1) strains, indicating that elevated ppGpp levels are not sufficient to elicit control of translation. In a relA^?Tic⁺ strain (NF162), or in a relA⁺Tic⁺ strain treated with rifampin, the ppGpp pool does not increase significantly after shift-down although translational initiation is reduced. Thus, an increase in the ppGpp pool is not necessary to control of translational initiation.