Frederick C. Neidhardt

The Regulation of RNA Synthesis in Bacteria

Publisher Summary The chapter discusses the regulation of RNA synthesis in bacteria. The regulation of RNA synthesis in bacterial cells has been explained in this chapter. Formation of these macromolecules seems to be geared in a precise and unique manner to the over-all protein-synthesizing potential of a cell in its particular environment. The synthesis of ribosomal ribonucleic acid (rRNA) is a variable fraction of a cells total biosynthetic activity, depending on the growth rate, so that the concentration of rRNA in a cell is a simple linear function of the over-all rate of protein synthesis during steady state growth. The conclusion that the constancy of the rate of protein synthesis calculated per unit of rRNA is the prime physiological function of this integration. The regulation seems to be achieved, by a reversible inhibition of the RNA-forming machinery of the cell, and most of the available evidence is consistent, with a model, in which amino acids reverse this inhibition, perhaps by combining with their respective sRNA. The shortcomings and limitations of the techniques most frequently is used in studying the regulation of macromolecule synthesis and pointed out the need for a different approach to this study is discussed in this chapter. The method shows promise of becoming an important tool for analyzing cellular regulatory devices.

Properties of a bacterial mutant lacking amino acid control of RNA synthesis

Abstract RNA synthesis has been studied in a strain of Escherichia coli which has lost amino acid control over this process. It was found that this strain is capable of a normal, physiological control of RNA synthesis under most conditions of balanced or unbalanced growth. It exhibits a normal correlation of RNA: protein ratio with growth rate in most media, and can halt RNA accumulation during adjustment to a poorer medim. In several conditions of cultivation, however, regulation of RNA synthesis by these cells breaks down, and they accumulate unusual amounts of RNA. Evidence is presented that all these conditions impose an amino acid restriction on the cells.

Characterization of the RNA formed under conditions of relaxed amino acid control in Escherichia coli.

Abstract When a strain of Escherichia coli with relaxed amino acid control overproduces RNA, the material made consists of 23-S, 16-S and 4-8-S RNA. When the overproduction occurs in the absence of protein synthesis, as following a shift down from amino acids to minimal medium, the pattern of accumulation is similar to that observed with normal cells inhibited by chloramphenicol. The 23-S and 16-S RNA are present as abnormal ribosomes, and proportionally less of these elements are formed than of s-RNA. At least part of the s-RNA appears to have amino acid accepting activity. When the overproduction occurs with some concomitant protein synthesis, as during adenosine inhibition, then the accumulated RNA appears as apparently normal 50-S, 30-S and soluble components in normal proportions. From these results it has been concluded that the single RC locus affects the normal synthesis of both ribosomal and soluble RNA during amino acid restriction.

Synthesis and inactivation of aminoacyl-transfer RNA synthetases during growth of Escherichia coli

Abstract A method has been developed to measure the rates of synthesis and of degradation of bacterial enzymes during growth by means of density labeling with deuterium to distinguish pre-existing from newly-made molecules. Use of this method has led to the discovery that some of the aminoacyl-transferRNA synthetases of Eschenchia coli are subject to high rates of irreversible inactivation during growth under amino acid restriction, particularly when such growth is intermittent. This fact explains why derepression of these enzymes during amino acid restricted growth has been difficult to observe. Measurement of the true de novo rate of formation of a number of synthetases reveals that each is regulated by a mechanism that normally maintains synthesis at less than 25 to 30% of its maximum rate, and that is capable of regulating this rate over a 10- to 50-fold range. The regulation bears a superficial resemblance to repression of biosynthetic enzymes since in both cases manipulation of the amino acid supply affects the rate of enzyme formation; nevertheless, there are conditions under which the regulation of synthetase formation is opposite to that of the biosynthetic enzymes.

Immunological and chemical studies of phenylalanyl sRNA synthetase from Escherichia coli

Use was made of preparations of phenylalanyl sRNA synthetase purified approximately 200-fold from a wild-type strain of Escherichia coli and from a mutant strain that possesses an altered form of this enzyme for which reactivity with p -fiuorophenylalanine is reduced. The purified preparations were antigenic in rabbits, inducing the formation of precipitating and enzyme-inhibiting antibodies. Inhibition of enzyme activity by rabbit antisera was quantitatively similar in the three assay systems commonly employed for aminoacyl sRNA synthetases. The antisera were equally inhibitory for activity in unfractionated extracts and for the purified enzyme. Enzymological analysis of the purified preparations demonstrated that the different reactivity of the mutant enzyme with p -fiuorophenylalanine was the result of a sixfold increase in K m and a 25-fold decrease in V max for this substrate. Nevertheless, the mutant enzyme appeared immunochemically indistinguishable from the wild-type enzyme. The purified preparations had only low levels of aminoacyl sRNA synthetase activity for other amino acids tested, and were homogeneous by immunochemical criteria. This apparent homogeneity after only a 200-fold purification implies that phenylalanyl sRNA synthetase comprises 0·5% of the total cell protein. The molecular weight of the enzyme was found to be approximately 160,000. A cell with a total protein content of 3 × 10 −7 μ g ought, therefore, to contain approximately 6000 molecules of phenylalanyl sRNA synthetase.

Regulation of formation of aminoacyl-ribonucleic acid synthetases in Escherichia coli

1. This study was undertaken to discover whether aminoacyl-soluble RNA synthetases in Escherichia coli are produced constitutively, or whether their synthesis is governed by a repression-like regulatory system. 2. The formation of phenylalanyl- and of isoleucyl-soluble RNA synthetases could be independently accelerated 2–3-fold over normal by growing cells under conditions in which phenylalanine or isoleucine, respectively, was growth-rate limiting. Moreover, restoration of an excess of the limiting amino acid caused a total, though temporary, repression of the elevated synthetase formation. 3. By the use of chloramphenicol and a rabbit antiserum to purified phenylalanyl-soluble RNA synthetase, evidence was obtained that derepression of this activity involves synthesis de novo of the enzyme protein. 4. The aminoacyl-soluble RNA synthetases for leucine and for histidine were not derepressed during growth limited by leucine or histidine. Isoleucyl-soluble RNA synthetase was not derepressed during growth limited by leucine. 5. These findings indicate that the rate of synthesis of some aminoacyl-soluble RNA synthetases is at least partially determined by repression. For these enzymes the repression can be lifted by restricting the amino acid supply. Aporepressors other than those regulating biosynthetic enzyme formation, however, seem to be involved.

Modification of valyl tRNA synthetase by bacteriophage in Escherichia coli

Abstract Infection of Escherichia coli with T4 bacteriophage is known to bring about the appearance of a new valyl tRNA synthetase activity distinguished by its behavior during hydroxylapatite fractionation, by its sedimentation in sucrose gradients and, in certain temperature-sensitive bacterial mutants, by its increased stability. Studies were made to determine the origin of this new activity. Its formation was accompanied at all times by a proportional decrease in the original valyl tRNA synthetase activity; 20 minutes after infection at 30 °C no original enzyme remained and no further increase in the phage-induced activity occurred. The addition of chloramphenicol at any time completely blocked both the gain of new and the loss of old activity. An increase of 2.6% in the buoyant density of valyl tRNA synthetase could be achieved by growth of the cells in 80% D 2 O medium, permitting resolution of heavy and light enzyme by equilibrium centrifugation with cesium chloride. By appropriate transfer of cells between deuterium-labeled and unlabeled media it was possible to demonstrate that the new enzyme activity brought about by phage infection in both mutant and wild cells consists of polypeptide chains synthesized prior to infection. From these results it was concluded that T4 infection causes a chloramphenicol-sensitive conversion of host valyl tRNA synthetase into a new, possibly dimeric form.

Effect of phage on amino acid activation.

Infection of Escherichia coli NP2 with T-even phage strains is known to cause the appearance of a new valyl tRNA synthetase activity. In the present work mutant hosts possessing altered activating enzymes for phenylalanine, glycine, or histidine were employed to detect possible phage-induced modifications in the translating systems for these amino acids. The results establish that T4 has an absolute requirement for the phenylalanyl tRNA synthetase of its host, probably in an unmodified form, and that this enzyme is responsible for the incorporation of most, if not all, phenylalanine residues into phage protein. Other data suggest, less rigorously, that a similar conclusion holds for glycine and histidine.

A gene of bacteriophage T4 controlling the modification of host valyl-tRNA synthetase.

Two hydroxylamine-induced mutants of bacteriophage T4 defective in modification of host valyl-tRNA synthetase have been isolated by assay of crude extracts for the activity that is characteristic of the wild-type virus. The mutations define a single gene that is situated between the rI and e genes on the T4 genetic map. This new gene is designated vs for valyl-tRNA synthetase. One of the mutations may be of the missense type since it results in the production of a valyl-tRNA synthetase activity that has unusual urea-inactivation properties. The other appears to be an amber mutation since the viral enzyme can only be found after infection of cells that are permissive for amber mutations. No differences in growth properties were found between wild type and amber mutant strains on the nonpermissive host. We conclude that the bacteriophage T4 valyl-tRNA synthetase is not essential for viability under prevailing laboratory conditions.