Robert F. Goldberger

Autogenous Regulation of Gene Expression The mechanism by which a protein directly controls expression of its own structural gene is described

A new term, autogenous regulation, is used to describe a phenomenon that is not a new discovery but rather is newly appreciated as a mechanism common to a number of systems in both prokaryotic and eukaryotic organisms. In this mechanism the product of a structural gene regulates expression of the operon in which that structural gene resides. In many (perhaps all) cases, the regulatory gene product has several functions, since it may act not only as a regulatory protein but also as an enzyme, structural protein, or antibody, for example. In a few cases, this protein is the multimeric allosteric enzyme that catalyzes the first step of a metabolic pathway, gearing together the two most important mechanisms for controlling the biosynthesis of metabolites in bacterial cells—feedback inhibition and repression. Autogenous regulation may provide a mechanism for amplification of gene expression (84); for severe and prolonged inactivation of gene expression (85); for buffering the response of structural genes to changes in the environment (45, 52); and for maintaining a constant intracellular concentration of a protein, independent of cell size or growth rate (86). Thus, autogenous regulation provides the cell with means for accomplishing a number of different regulatory tasks, each suited to better satisfying the needs of the organism for its survival.

[147] Enzymes and intermediates of histidine biosynthesis in Salmonella typhimurium

Publisher Summary This chapter focuses on enzymes and intermediates of histidine biosynthesis in Salmonella typhimurium. A series of 10 enzymes in Salmonella typhimurium mediates the conversion of phosphoribosyl-pyrophosphate and ATP to histidine. Mutants lacking all 10 enzymes require only histidine for growth; therefore this pathway has no branches giving rise to other essential metabolites. The intermediates of the pathway have been isolated and characterized and assays for all 10 enzymes have been devised. The PR-AIC is converted back to ATP by way of the purine biosynthetic pathway. The histidine operon on the S. typhimurium (and probably on the Escherichia coli ) chromosome is a cluster of the structural genes for the histidine biosynthetic enzymes. The enzymes are always purified either from cells grown under conditions of derepression or from strains containing a constitutive mutation. Several of the assays for the histidine biosynthetic enzymes are followed spectrophotometrically. These assays generally have a total volume of 0.3 ml in cuvettes of 1-cm light path and a total capacity of 1 ml. Purification of the dehydratase-phosphatase complex has proved to be exceedingly difficult because the enzyme appears to undergo multiple and still unpredictable aggregation disaggregation reactions.

Regulation of Histidine Biosynthesis in Salmonella Typhimurium

Publisher Summary Regulation of histidine biosynthesis in Salmonella typhimurium has been the subject of intensive investigation in a number of laboratories. These studies have brought to light some of the basic phenomena of molecular biology. The pathway for histidine biosynthesis in Salmonella typhimurium consists of a series of 10 reactions, each catalyzed by a specific enzyme. The rate at which these enzymes convert adenosine triphosphate (ATP) and 5-phosphoribosyl 1-pyrophosphate (PRPP) into the amino acid, histidine, is regulated by feedback inhibition: the activity of the enzyme that catalyzes the first step of the pathway is inhibited by the end product, histidine. The structural genes for the 10 enzymes of histidine biosynthesis, together with an operator gene, are clustered on the Salmonella chromosome in the region known as the histidine operon. The rate at which the information encoded in the operon is utilized for synthesis of the enzymes is under repression control. The operon functions as a unit in response to the level of histidine available to the organism. Under the conditions in which ample histidine is available, as in the wild-type organism, the operon is utilized at a low rate, maintaining a basal intracellular level of the histidine enzymes; when the availability of histidine limits the growth rate of the organism, the operon is utilized at a higher rate, causing an increase in the intracellular concentrations of the enzymes, up to a level approximately 20-fold higher than the basal level.

Regulation of Gene Expression in Prokaryotic Organisms

Publisher Summary This chapter focuses on the regulation at the level of gene expression. The structural genes for what the transcription frequency can be regulated have been the subject of intensive investigation over the past two decades. There are many steps in the process of transcription of a given gene that finally results in a change, in a specific cellular function; some of these steps that are capable of being regulated are defined. Binding of ribonucleic acid (RNA) polymerase to the promoter involves a number of different proteins that modify the activity and specificity of the polymerase, allowing gene expression to be regulated. The effects of the regulatory proteins may be negative, restricting transcription, or positive, facilitating transcription. The activities of the regulatory proteins are themselves regulated by specific small molecules that communicate the metabolic needs of the cell to the sites at which gene expression is controlled. Gene expression could be modulated by alterations in the stability of the mRNA, in the frequency with what specific portions of a polycistronic mRNA are translated, in the availability of the various species of tRNA or their synthesis, or in any of the many catalytic events involved in the translation process. The activity of a protein may be modified by specific small molecules through noncovalent interactions, such as those involved in “feedback” inhibition, or through covalent changes, such as adenylylation. By describing the mechanisms of modulating gene expression, the basic principles involved and to indicate how complex is the total story of gene regulation, and how extensively interlocking are its parts are elucidated.

Studies on the size of the messenger-RNA transcribed from the histidine operon during simultaneous and sequential derepression

The techniques of double isotope labeling and specific hybridization have been used to determine the size of the mRNA transcribed from the histidine operon in Salmonella typhimurium under conditions of derepression. The results obtained by both techniques agree with the previous results of Martin3 which demonstrated that, in a constitutive mutant, the histidine mRNA is polycistronic. Previous studies revealed that derepression of the enzymes for histidine biosynthesis may proceed by either of two modes1,2. One mode is characterized by simultaneous derepression of all the enzymes, whereas the other mode is characterized by the derepression of the enzymes in a temporal sequence which corresponds with the positional sequence of genes in the histidine operon. Since the present studies show that the mRNA transcribed from the histidine operon is always polycistronic, regardless of the mode of derepression, we may rule out the possibility that the difference between the two modes reflects any difference in the size of the histidine mRNA. Rather, the difference must lie in the manner in which the polycistronic histidine mRNA is translated.

The Effect of Estrogen on Gene Expression in Avian Liver

About four years ago, we turned our attention from regulation of gene expression in prokaryotes and began to search for a system that would tell us something about regulation in eukaryotic cells. We were guided in this search by the following considerations. First, we did not want to work with a system in which cellular differentiation was itself an integral part of the regulatory response. We felt that we could obtain more clear-cut information if we could study a response in a tissue that was already fully differentiated and in which the regulatory response did not require DNA synthesis. We recognized that a hormonally responsive system would be the easiest to manipulate, and for practical reasons we wanted a system in which the hormonal response was of very great magnitude. And as long as were asking, we thought we might as well put in a request that the tissue we study also contain a whole group of genes that are hormonally responsive, so that we would be able to study not only the effect of hormone on a specific gene but also the mechanisms that coordinate the expression of the hormone-responsive domain of the tissue. In addition, we wanted our hypothetical tissue to have a gene expressed at a high level that is not responsive to hormonal stimulation. Such a gene would provide an ideal control for in vitro transcription studies, where one must show that the specificity of hormonal responsiveness reflects that of the tissue in vivo.

A new and highly sensitive assay for the ATP phosphoribosyltransferase that catalyzes the first step of histidine biosynthesis

Abstract A new method is described for assaying the ATP phosphoribosyltransferase (EC 2.4.2.17) that catalyzes the first step of histidine biosynthesis in bacterial cells. This is a highly sensitive radioassay, capable of detecting fewer than 10 9 molecules of enzyme. In contrast to the previously available assay, it can be carried out in the presence of other molecules that absorb uv light. The assay may find useful application in studies on the in vitro synthesis of the enzymes for histidine biosynthesis and in studies on the various aggregation states of the enzyme.

Strategies of Genetic Regulation in Prokaryotes

One of the most striking characteristics of living systems is that they function in an orderly manner despite their high degree of complexity. One workable definition of regulation, in fact, is the set of mechanisms that allows organisms to maintain this orderly functioning. It is important to realize, however, that regulation was not superimposed upon living systems; orderly processes are simply more successful than are disorderly ones, and therefore tend to be preserved through the evolutionary process by conferring advantages upon organisms that possess them. The thousands of chemical reactions occurring in cells are controlled by regulatory mechanisms that operate at many different levels. This introductory chapter focuses on those that operate at the level of gene expression and will introduce some of the strategies of genetic regulation that have evolved in prokaryotic organisms. Scanning the table of contents of this brief essay should suffice to tell the reader that a very general overview is in store for him. The renaissance in biological research that occurred in the last 25 years has been due mostly to the exciting studies concerning genetic regulation in prokaryotes. I have tried to abstract from those studies the most important basic principles they illustrate and to organize into a few generalizations the enormous body of data they have produced. I believe it is these principles and generalizations with which the reader will need to arm himself before proceeding further into this volume. It is to be hoped that the necessarily simplistic view of regulation they provide will be preferable to the bewilderment that so often results from exhaustive reviews that include the details of many specific regulated systems.

EFFECT OF ESTROGEN ON GENE EXPRESSION: VITELLOGENIN SYNTHESIS MAY BE REGULATED AT THE LEVEL OF BOTH TRANSCRIPTION AND TRANSLATION

Administration of a single dose of estrogen to a rooster results in de novo synthesis of vitellogenin mRNA and vitellogenin in the liver. When the estrogen response has apparently ceased and vitellogenin is no longer detectable in the liver or in the plasma (post-estrogen-serum-negative state) vitellogenin mRNA persists in appreciable amounts in the liver of the rooster. This mRNA has the same size as vitellogenin mRNA from stimulated roosters. However, this vitellogenin mRNA is no longer associated with polysomes. The possible role of this persisting vitellogenin mRNA in the greater rapidity and magnitude of the response to a subsequent estrogen stimulation will be discussed.

Kontrollmechanismen lebender Systeme

Zu den auffalligsten Eigenschaften lebender Systeme gehort deren Fahigkeit, geordnete und sinnvolle Funktionen auszufuhren. Die hierbei wirksamen Kontroll- und Regelvorgange sollen in diesem Kapitel beschrieben werden. Ein Teil dieser Kontrollmechanismen lauft innerhalb der Zellen ab, ein anderer Teil verlauft uber Wechselwirkungen zwischen verschiedenen Zellen. Dabei mus man voraussetzen, das sowohl Organisation als auch Regulation keine kunstlichen, von ausen dem lebenden Organismus „auf gesetzt en“Eigenschaften sind, sondern das sie dem lebenden System als biochemische Charakteristika innewohnen. Die Natur verwendet dabei die Regulationsmechanismen nicht, um biochemische Prozesse zu ordnen, sondern geordnete Reaktionen sind gegenuber den unkontrollierten Prozessen vorteilhafter und organisierte Strukturen waren im Verlaufe der Evolution stets bevorzugt. Die Selektion der belebten Natur erfolgte nicht auf Grund kluger oder schopferischer Entwicklungen, sondern auf der Grundlage des chemisch gunstigsten Prozesses.