Metabolite Analogs as Genetic and Biochemical Probes

METABOLITE ANALOGS AS GENETIC AND BIOCHEMICAL PROBES H. E. Umbarger Department of Biological Sciences, Purdue University, Lofayette, Indiana

I. 11. 111. IV. V. VI. VII.

Introduction . . . . . . . . . Tryptophan Analogs . . . . . . . Tyrosine Analogs . . . . . . . Phenylalanine Analogs . . . . . . Histidine Analogs. . . . . . . . Analogs of Isoleucine, Valiie, and Leucine Outlook and Recommendations. . . . References . . . . . . . . .

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119 122 126 127 128 131 135 136

I. Introduction

I n the course of their fine-structure studies of the genetic material in Salmonella typhimurium, Demerec and colleagues were greatly intrigued by their discovery that genes controlling related biochemical steps were often linked. At the time, Demerec (1955) suggested that this nonrandom gene distribution, which had not been noted with fungi, was not a matter of chance but a result of some strong evolutionary advantage, perhaps peculiar to bacteria. What that advantage was probably escaped him and it cannot yet be fully explained. On the basis of some of the studies that stemmed from his pioneering experiments, however, we can cite one very distinct advantage that the clustering of related genes provided. That advantage was recognized when it was discovered that the expression of gene clusters could be regulated together, presumably through another genetic element, the operator (Ames and Garry, 1959; Jacob and Monod, 1961). Clearly, the role of a regulatory element that controls the expression of a gene cluster is to “recognize” some specific signal generated within the cytoplasm that allows the gene cluster to function a t varying rates. In the case of a cluster of genes involved in amino acid biosynthesis, a signal to “turn off” or repress gene function is generated when the amino acid is supplied in the medium. Since it is quite unlikely that the DNA of the postulated regulatory element or operator could directly 119

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recognize an amino acid, some additional regulatory element must be postulated to mediate the interaction between excess amino acid and the DNA of the operator. In the model of Jacob and Monod (1961), the repressor has been postulated to play this mediating role. Proposing this second element leads to the assumption that a second regulatory gene must exist. Since regulatory elements involved in repression studies will not give the phenotype of growth factor requirement upon loss of function, genetic techniques different from those employed in the isolation of auxotrophic or carbohydrate nonfermenting mutants had to be devised. To date, the most successful probe for the analysis of these gene-gene interactions leading to control of gene functions is the use of metabolite analogs that are inhibitory to cell growth. Many examples serve to illustrate the way in which this approach has been successful, but it should be noted that the approach is far from being fully exploited. One of the striking facts to emerge from this kind of an approach is that the generation of repression signals can be far more complex than the two-element model originally postulated by Jacob and Monod (1961).In other words, there can be several elements involved in end product recognition, suggesting that a series of reactions might be involved. It might be of some interest to review some of the patterns that appear to be emerging from these ongoing attempts in several laboratories to ana1yz;e the mechanisms of gene expression. Before doing so, however, it might be well t o define some terms and to outline what this writer looks upon as the 1970 version of the Jacob-Monod model in its pure form. As postulated in 1961, expression of a cluster of structural genes, and presumably that of a single structural gene as well, is controlled by a repression recognition element, the operator. Transcription of the structural genes occurs only if the operator region is not bound to the repressor, which is itself the product of a regulatory gene. Since the regulatory gene functions via a cytoplasmic product, the location with respect to the cluster is left unspecified. While the original model did not specify the nature of the repressor, the two repressors that have been isolated are, in fact, protein (Gilbert and Muller-Hill, 1966; Ptashne, 1967). The general properties of these repressors will be taken as the criteria for a repressor in the strict sense of the word: a protein that binds specifically to a fragment of doublestranded DNA containing a specific repression recognition (operator) region (Gilbert and Muller-Hill, 1967). Thus, genetic evidence for an element required for generation of a repression signs1 is no longer sufficient evidence to postulate a repressor. The gene cluster with its adjacent operator was originally considered

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to be the operon. A feature of the operon was that, even though it might have been composed of several structural genes, it functioned as a single unit of expression with presumably transcription of the genetic information beginning at the first gene in the cluster and continuing through the last. Unsettled even today is the question whether the operator is transcribed and, if transcribed, whether it is translated. The failure to find a fragment of the z gene of the lac operon in Escherichia coli corresponding to the operator region (Bhorjee et al., 1969) does not appear to be relevant since it is inferred that a t least a portion of the N-terminal region has been removed (e.g., p-galactosidase does not contain an N-formylmethionine group). That the operator might indeed be transcribed can be concluded from the demonstration of still another element, the promoter, which appears to be the point at which RNA polymerase begins its transcription. I n the case of the lac operon, for which the evidence for a promoter is strongest, the promoter is also now believed to be an element of control in that it includes the recognition region for catabolite repression or perhaps more precisely for the cyclic 2’,3’-adenosine monophosphate (CAMP)-dependent protein. Whereas the repressor is a negative control element, the CAMP-dependent protein is a positive control element (Emmer et al., 1970; Beckwith, 1970). The lac promoter thus has two postulated functions that have not been separated structurally: a point of transcription initiation and a recognition site for a positive control element. Whether these two functions will be necessary properties of all promoter regions remains to be seen. That two kinds of recognition element have evolved in the case of the lac operon can be interpreted in terms of the physiological advantage that was achieved. Although the original Jacob-Monod model did not postulate either a promoter or a positive control element, it does seem fair to retain both of these elements in the 1970 version of the model. Therefore, in this paper, the model will be considered to involve the operon consisting of the promoter, operator, and structural gene(s), in that order, and presumably, but not necessarily, two regulatory genes that are themselves structural genes for a positive and a negative control element. A minor departure, such as a series of reactions required to convert some small molecule to the metabolite that binds to the repressor or to the positive control element, would also be in accord with the model. At this point, the question of what regions are transcribed in the mRNA or translated into polypeptide can remain unspecified. Also permissible in the interpretation of the model being followed here would be an operon without an operator. It would not be difficult to imagine a system in which a repression recognition region was not

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present and the entire control of the operon resided in the kind of specificity exhibited by a positive control element. (The CAMPdependent protein appears to have a very broad specificity affecting a variety of, if not most, catabolic enzymes.) Thus, a highly specific positive control element analogous, if not homologous, to the CAMPdependent protein might eliminate the need for a repressor. Conversely, a “low-level” promoter, which recognized either no protein analogous to the CAMP-dependent protein or one in which its activity did not vary with the environment, might account for formation of proteins that are always found in fixed levels, if any such do exist (Pardee and Beckwith, 1963). For the purposes of this paper, it will be assumed that, none of the systems that will be considered here have yet been shown to fulfill the necessary features of the Jacob-Monod model. Specifically, the missing evidence in each case is failure to demonstrate a repressor, i.e., a protein binding to the operator. Therefore, general terms, noncommittal with respect to mechanism, will be preferred to the terms operator and repressor which now refer to very precisely defined physical entities. Thus, until binding to a specific repressor is demonstrated, the terms “repression recognition element” or “induction recognition element,” will be preferred to the term “operator.” (The genetic designation for such elements with the symbol 0 as in his 0 and leu 0 is heartily endorsed, however.) Similarly, “repression signal” or “induction signal” will be preferred to “repressor.” (The apparent complexity of some of the systems generating such signals is a strong justification for this operational terminology.) The model employed here is thus not intended as an alternative to the Jacob-Monod model but as a more general scheme that encompasses alternatives to the model. Furthermore, no restrictions will be made with respect to control at the transcriptional or translational level. II. Tryptophan Analogs

The technique of using an analog as a genetic and biochemical probe in bacteria had its origin with the isolation by Cohen and Jacob (1959) of 5-methyltryptophan-resistantstrains of E. coli. These workers demonstrated that the mutated genetic locus ( t r p R ) was distant from the tryptophan structural genes on the E. coli chromosome (Cohen and Jacob, 1959). It will be recalled that the trp R lesions resulted in nonrepressible synthesis of the tryptophan biosynthetic enzymes. The existence of such mutants was readily interpreted by a version of the

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Jacob-Monod model in which the repressor (in this case the trp R gene product) was active only when excess endproduct was present (in this case tryptophan). Today, the trp R gene is usually referred to as the structural gene for the tryptophan repressor (Taylor, 1970). In the more general terminology recommended here, the trp R gene should be looked upon for the present merely as an element required for generating the signal, “tryptophan excess.” One does not know whether it is the only element that is required or not.” The 5-methyl analog of tryptophan was also employed by Moyed (1960) who found an entirely different kind of 5-methyltryptophanresistant mutant. This mutant was shown to have an anthranilate synthetase and a phosphoribosyl anthranilate transferase that were resistant to inhibition by tryptophan or by 5-methyltryptophan. Surprisingly, these mutants were also derepressed with respect to the tryptophan biosynthetic enzymes. Mutants of this type have subsequently been studied in both E. coli and X. typhirnurium and have thus far been only partially explained (Somerville and Yanofsky, 1965; Cordaro and Balbinder, 1967). [That two enzymatic steps were sensitive t o end product inhibition is explained by the fact that the synthetase and the transferase exist as a complex and that the inhibition of the transferase activity is due to binding of tryptophan to the synthetase (It0 and Yanofsky, 1966, 1969). It should be noted, however, that the transferase was also sensitive to tryptophan when complexed with a tryptophaninsensitive anthranilate synthetase. Clearly, the tryptophan site on the anthranilate synthetase had not been destroyed but only its interaction with the catalytic site.] The genetic study of these same mutants demonstrated that most had lesions in the “initial” region (i.e. near the beginning of the gene cluster which, in fact, begins with the anthranilate synthetase structural gene) (Somerville and Yanofsky, 1965; Cordaro and Balbinder, 1967). The simultaneous development of derepression and loss of end product sensitivity exhibited by these mutants thus raised two questions: ( a ) Was the repression recognition site (operator) part of the first structural gene in the operon? and ( b ) were end product sensitivity and repression inextricably linked in function? An affirmative answer to the first question would have been rather readily accommodated by the Jacob-Monod *It is ironical that the tryptophan biosynthetic system is one of the few in which a test for a repressor is possible. Preparations of trp DNA with and without the repression recognition region ( t r p 0) are readily available and the trp R gene has been shown to be the structural gene for a protein (Matsushiro e t ~ l . , 1965; Ito e t al., 1969). However, it is this writer’s impression that attempts to find a trp 0-binding protein in t r p R+ cells have been unsuccessful to date.

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model, whereas an affirmative answer to the second would have been strong support for an alternative that involved control at the level of translation and more specifically the binding of end product and the first enzyme in the sequence. Indeed, a very specific model to account for translational control of gene expression was proposed several years ago by Cline and Bock (1966) based on the assumption that end product inhibition and repression were linked and on the occurrence of several other observations that were not immediately predicted by the Jacob-Monod model. For the most part, however, that model was not uniquely supported by the observations that were cited. While the model proposed by Cline and Bock (1966) was perhaps much more explicit than the data would justify, there are a number of observations made on apparent relationships of end product-sensitive enzymes to repression that are difficult to account for at present by a repressor-operator model. Nevertheless, in no case do these examples point so strongly to alternative models that an ad hoc accommodation with the repressor-operator model is ruled out. Two quite different classes of 5-methyltryptophan-resistant mutants of E . coli have more recently been identified by Hiraga and co-workers (Hiraga, 1969; Hiraga et al., 1968). One of these classes was particularly important for it provided the first genetic evidence in this organism for a repression recognition region ( t r p 0). An operator region had been inferred to exist but, as well as this reviewer can determine, the inference was from analogy with the Jacob-Monod model and from the direction of polarity of the genetic information (Yanofsky and Ito, 1966). There were, however, defective @80 phages that carried varying portions of the trp gene cluster in their genomes (Matsushiro e t al., 1965). Two of these that carried all five trp cistrons differed from each other in that one was repressible and the other was not. The repression recognition element was therefore assumed to have been severed in the one case. This kind of observation, however, does not provide as rigorous evidence for a repression recognition element as does the O++ 0" type of mutation first demonstrated for the lac operon (Jacob and Monod, 1959). The other class of 5-methyltryptophan-resistant mutants has not as yet been explained biochemically. They affect a region of the chromosome (designated mtr) that is remote from t r p R , the t r p structural gene cluster, and trp S, the tryptophanyl-tRNA synthetase structural gene (Hiraga et al., 1968). At the present time there is no evidence that these mutations in any way affect expression of the trp operon. It is of interest that the genetic studies of Hiraga (1969) with the trp Oc mutant suggested that the trp 0 region was about one fourth as long as the entire t r p operon. If the t r p 0 region were transcribed

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(as might be inferred from the current idea of the role of the promoter as found in the lac system), it would constitute a significant fraction of the mRNA hybridizable with trp DNA after derepression of the trp operon. However, Imamoto (1969) has shown that a mutant bearing a deletion of all but about one eighth of the operator-proximal portion of the trp operon has retained the repression recognition site. Measurement of trp mRNA formed by this mutant indicates that if trp 0 is transcribed, it cannot account for more than 3% of the trp operon. It may be, of course, that the size estimate of the trp 0 region made by Hiraga was in error because some unknown factor perturbed the recombination frequency or UV-inactivation measurements. Evidence for a repression recognition region preceding the trp structural genes has also been obtained for X. typhi1nurium by Callahan et a2. (1970) who isolated derepressed mutants with lesions in the postulated trp 0 region that were resistant to 5-methyltryptophan and t o 6-fluorotryptophan. They also marshalled evidence that the promoter element deleted in some tryptophan auxotrophs described by Margolin and Bauerle (1966) was adjacent to the repression recognition region and distal to the structural genes similar to the arrangement in the lac system of E . coli. Held and Smith (1970a)b) have analyzed the effect of another analog of tryptophan, 7-methyltryptophan, as well as two analogs of tryptophan precursors, 3-methylanthranilatc and 7-methylindole1 which can be converted to 7-methyltrytophan. They showed that all three compounds caused a derepression of the trp operon in wild type E. coli which they attributed to tryptophan starvation resulting from inhibition of anthranilate synthetase by 7-methyl-tryptophan. Two classes of 3-methylanthranilate-resistant mutants were found-one class with lesions closely linked to the trp gene cluster, and a second class with lesions close to aroG, the structural gene for the phenylalanine-sensitive DAHP synthetase. The former contained anthranilate synthetases that were not sensitive to end product inhibition. These mutants were also resistant to derepression of the t r p genes by 7-methyltryptophan. It was concluded on this basis that the primary cause of derepression by 3-methylanthranilate was the inhibition of anthranilate synthetase by the endogenously formed 7-methyltyptophan. As will be discussed later, however, it may not always be possible to identify the target site of an analog as the site of analog resistance. The 3-methylanthranilate-resistant mutant of the second class was interesting not because it pointed to an unrecognized genetic region but because it revealed a previously unrecognized metabolic relationship. This mutant had a DAHP synthetase that was resistant to inhibition

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by phenylalanine and was shown to overproduce chorismate (Held and Smith, 1970a). Since the latter is the substrate that competitively overcomes 7-methyltryptophan inhibition of anthranilate synthetase, it would appear that it was overproduction of substrate that was responsible for resistance. While overproduction of a competitive substrate had been anticipated as a mechanism of analog resistance (Davis, 19571, one cannot a priori assume that a sufficiently elevated pool of the substrate can, in fact, be achieved. The mutant in this case suggests that the chorismate pool can be so elevated. The second class of mutant is, of course, one that would account for resistance if the postulated target site were the actual one. However, it would also account for resistance if what was really necessary was increased metabolite flow over the tryptophan biosynthetic pathway and an increased internal tryptophan pool. In part, the use of analogs has been used to support the argument that the activation of tryptophan is not necessary for the generation of the repression signal recognized by the trp 0 region. That there might be some role of tryptophanyl-tRNA synthetase in repression had been indicated by the demonstration that a mutant with an altered synthetase exhibited an altered pattern of repression and derepression of the t r p operon (Kano et al., 1968; Hiraga et al., 1967). That this involvement did not include the charging of tRNA with tryptophan, however, was suggested by the observations on three analogs of tryptophan: 7-azatryptophan, 4-methyltryptophan1 and 6-methyltryptophan (Mosteller and Yanafsky, 1970). The first two could be incorporated into protein, whereas the third could not. In contrast, the first was unable to repress the formation of specifically hybridizable t r p mRNA, whereas the second and third could. 111. Tyrosine Analogs

Quite a number of tyrosine analogues have been tested in microorganisms, but only a few have been subjected to the kind of genetic and biochemical analysis that is being considered here. Among these is 4-fluorophenyla1aninel an analog shown years ago by Munier and Cohen (1956) to be incorporated into E. coli protein. More recently, 4-fluorophenylalanine-resistantmutants of S. typhimurium have been examined in which the lesions are closely linked to structural genes for two enzymes that are coordinately derepressed in these mutants, the tyrosine-repressed DAHP synthetase and prephenate dehydrogenase (Gollub and Sprinson, 1969). While other, unlinked tyrosine repressible gene products have not been examined, it may be that, in this case,

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a specific repression recognition region has undergone an 0' + 0"type mutation. Another kind of mutation has been found in E. coli mutants resistant to 4-aminophenylalanine (Wallace and Pitt.ard, 1969). I n these, the mutated gene, tyr R, also causes derepression of the aroF or tyr A genes but is unlinked to them. In addition, transaminase A, an enzyme for which the structural gene has not been located, is derepressed. At present, in the operational terms employed here, tyr R is the gene for some element (not necessarily a repressor) required for generating the tyrosine excess signal. Still another analog of tyrosine, 2-aminotyrosine, has been employed with E. coli strain 9723 as a biochemical probe but not as a genetic probe. Sloane and Smith (1968), in an attempt to explain the reversal of the inhibitory effect of this tyrosine analog by p-hydroxymethylphenylalanine but not by phenylalanine, obtained isotopic evidence that p-hydroxymethylphenylalaninecould be converted to both phenylalanine and tyrosine. Further analysis yielded evidence that some phenylalanine could be converted to p-hydroxymethylphenylalanine which, in turn, could be an intermediate in the low-level conversion of phenylalanine to tyrosine that these workers observed. Whether this route is unique to certain strains of E. coli and what its relationship is to the well-known route to tyrosine via prephenate remains an open question. Interestingly, however, it is a route that, on the basis of mutant methodology with the more commonly used strains of E. coli and S. typhimurium, has been eliminated as a significant one. Finally, D-tyrosine has been employed as an analog of L-tyrosine in Bacillus subtilis by Champney and Jensen (1969). A resistant mutant was isolated in which the gene lesion was in the structural gene for prephenate dehydrogenase, which, in the mutant, differed from that in the parent in being resistant to end product inhibition by tyrosine. Such findings are extremely important, for the parallel observation that such mutants excrete L-tyrosine provides evidence that the end product sensitivity observed in vitro is indeed physiologically significant. The studies did not reveal, as these workers correctly interpreted, whether inhibition of prephenate dehydrogenase or some other enzyme by D-tyrosine was the primary cause of the growth inhibition. This principle is sometimes forgotten in the interpretation of analog-resistance studies. IV. Phenylalanine Analogs

An often used analog of phenylalanine is p-2-thienylalanine. One of the first uses of it as a biochemical probe was reported by Ezekiel (1965)

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who showed that it inhibited the phenylalanine-repressible DAHP synthetase as well as phenylalanyl-tRNA synthetase. A resistant mutant exhibited what was very probably an altered phenylalanine repressible DAHP synthetase. The genetic analysis of this strain does not appear to have been completed, however. In B. subtilis, the site of end product inhibition by phenylalanine is prephenate dehydratase. Coates and Nester (1967) have isolated two types of p12-thienylalanine-resistantmutants in which the properties of this enzyme are altered. I n one type, the enzyme is insensitive to phenylalanine and in the other the enzyme is actually stimulated by phenylalanine. Genetic analysis verified that the structural gene for the enzyme was modified. Phenylalanine and tyrosine provide two very striking examples of amino acids for which analogs have certainly been underexploited. There is such a wide variety of analogs for these amino acids (for a list of some, see Shive and Skinner, 1963) that have not a t all been subjected to a combined biochemical and genetic analysis. V. Histidine Analogs

Probably the best example of the utility of amino acid analogs as biochemical and genetic probes is to be found in the studies conducted by Hartman and Ames and their colleagues. That the genetic analysis has been so successful is due to a great extent to the very extensive genetic mapping studies that have been conducted with the histidine auxotrophs of S. typhimurium. The introduction of histidine analogs into serious microbial studies is due to Moyed (1961), who employed 2-thiazolealanine as an inhibitor of E . coli. Moyed discovered that this compound was an inhibitor of the first enzyme in the pathway to histidine. At low levels of thiazolealanine, growth inhibition was, however, only transient, since the limitation of histidine caused the derepression of all the histidine biosynthetic enzymes including the sensitive one. This mechanism, termed by Moyed “induced phenotypic resistance,” has been found many times since with analogs. It will not occur with all analogs, however, because some will prevent protein synthesis (and hence the derepression necessary to overcome the inhibition) or may, in fact, be incorporated into protein but yield inactive or “false” proteins. Another possible reason for the absence of induced phenotypic resistance is that derepression does not occur because the analog mimics the effect of the natural amino acid as a repressor. This mechanism has often been postulated but is difficult

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to distinguish from “false protein” formation. I n only a few cases does it appear that this distinction has been achieved. One of the most strongly supported cases for an analog mimicking the effect of an end product as a repressor is that of 1,2,4-triazolealanine1 studied by Levin and Hartman (1963). Triazolealanine is a histidine analog that is incorporated into protein to yield, in at least some cases, biologically inactive protein. These workers examined several histidine biosynthetic enzymes formed in the presence of triazolealanine by a mutant that had completely lost the repression recognition region as well as part of the gene for the first enzyme in the pathway but could express the other his genes, (i.e., a his Oc G mutant). It was possible to demonstrate that active histidinol phosphate phosphatase and imidszole acetolphosphate transaminase were formed in the presence of triazolealanine, whereas an active histidinol dehydrogenase was not formed. However, when a leaky mutant that, in the absence of histidine, formed both enzymes a t a derepressed rate was given either triazolealanine or histidine, both enzymes were repressed. Unless, by some peculiar mechanism, these two enzymes were formed in the deletion mutant because histidine released by protein turnover was incorporated into these two enzymes in preference to the analog, one must conclude that triazolealanine can indeed be used to mimic a “histidine excess” signal. The first extensive genetic analysis of mutants resistant to a histidine analog was that performed by Sheppard (1964) with S. typhimurium mutants resistant to triazolealanine, the analog shown by Moyed to act as a “false feedback” inhibitor. Sheppard noted that most of the mutants had lesions in a particular region of the his G gene which specified the structure of the first enzyme in the pathway to histidine. In such mutants, the enzyme was refractory to inhibition by both histidine and thiazolealanine. While this region is not necessarily that which specifies the histidine binding site, it does apparently specify a sequence in the protein that is a t least important in end-product sensitivity. The region is also of special interest since mutations occurred here that appeared to result in the generation of new transcription initiation points (promoters?) in a strain that has lost by deletion both the repression recognition region as well as the postulated promoter (St. Pierre, 1968; Ames et al., 1963). Furthermore, some of the reinitiation mutations in this region, when transferred to a strain in which the rest of the his operon was intact, led t o cold-sensitive strains that were super-sensitive to inhibition by histidine similar to the E. coli mutants described by O’Donovan and Ingraham (1965). The genetic analysis of S. typhiinurium mutants in which regulation of formation of the histidine biosynthetic enzymes was altered, has been

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more spectacular than the analysis of the mutants with altered end product sensitivity. These studies have revealed that regulation in this system is indeed more complex than was a t first realized. Because triazolealanine prevents derepression, it will prevent the growth of organisms that depend upon derepression of the histidine gene cluster for growth. Examples would be bradytrophs (“leaky” mutants in which one enzyme in the pathway was only partially active) and cells growing in the presence of 1,2,4-amino triazole, an inhibitor of imidazoleglycerol phosphate dehydrase. In such cases, derepression was required for an adequate rate of histidine biosynthesis. The first gene to be recognized by lesions leading to derepression was designated his R , undoubtedly in anticlipation that it was the gene for the repressor of the Jacob-Monod model (Roth and Hartman, 1965). Subsequently, it was found that the derepressed mutants contained lesions in any of several additional loci, which were designated his S, T , U , and W (Roth et al., 1966, Anthn, 1968). Examination of these revealed that the his S mutants had lesions in the structural gene for histidyl-tRNA synthetase, while his U , his W ,and the earlier recognized his R mutants had reduced levels of histidine acceptor tRNA (An&, 1968; Roth and Ames, 1966; Silbert et al., 1966). While there might be reason to suspect that his R, W , and U are structural genes for histidine acceptor tRNA, there is no evidence that there are three distinct species. It is not a t all clear how these genes might affect the quantity of a single species of tRNA. The possibility might be considered that they are multiple copies of a gene that does not function very effectively so that all three are required for the full quota of histidine acceptor tRNA.” The his T gene is not yet explained but it does appear to be the structural gene for a protein (B. N. Ames, personal communication). What can be concluded at this point is that activation of histidine by the histidyl-tRNA synthetase and an intact his T gene are essential for the generation of the excess histidine signal. That a deficiency of the corresponding tRNA itself also prevents the generation of the signal suggests that histidyl-tRNA itself is directly on the pathway to the repression signal. Indeed, a direct involvement of these recognized elements would still be compatible with the Jacob-Monod repressor-operator model. Alternatives are possible, however, including some in which histidine acceptor tRNA plays an indirect role. One alternative is based on the observations of Goldberger and his associates (Kovach et al., 1969) that, in certain mutants with an initial enzyme in the pathway

* More-recent observations indicate that the levels of histidine acceptor tRNA in his U and his W strains are normal. Only his R mutants exhibit reduced levels of histidine acceptor tRNA.

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that is histidine-insensitive, triazolealanine cannot repress the histidine biosynthetic enzymes, and that, with histidine itself, the pattern of repression is markedly altered. Their further observations that the initial enzyme binds histidyl-tRNA (though not completely specifically) had induced them to invoke some not-yet clear role for the first enzyme in the generation of the repression signal (Kovach et al., 1970). Certainly, the studies currently in progress with this system are worthy of our continued attention. Another analog of histidine, D-a-hydrazinoimidazolepropionic acid (HIPA) has been useful in the study of the active transport systems for histidine in 8. typhimurium (Ames and Roth, 1968). HIPA is an inhibitor of growth and, while the primary site of its inhibitory effect is presumably not active transport, apparently the most effective way for the organism to develop resistance is to prevent the uptake of the analog. The analysis with HIPA and a variety of other analogs indicated that histidine was concentrated in the cell by means of two uptake systems, one highly specific for histidine and histidine analogs and another for aromatic amino acids or histidine. More recently, studies involving utilization of D-histidine in place of L-histidine have revealed genetic evidence for two histidine-binding proteins in addition to the component earlier referred to as a “permease” (Ames and Lever, 1970). This example is only one of many that could be cited in which analogs have been used to select for mutants with altered uptake of the analog as well as the natural product. Clearly, analogs should be useful in helping to unravel the complex system that the word “permease” conceals. VI. Analogs of Isoleucine, Valine, and Leucine

As might be expected from the similarity in structure of isoleucine, valine, and leucine, these amino acids have sometimes functioned as analogs of each other. Indeed, one of the earliest examples of “amino acid imbalance” was observed with these amino acids by Gladstone (1939) using B . anthracis. The well-known inhibition of the growth of the K-12 strain of E. coli by valine, and its reversal by isoleucine, provided the subject of the studies conducted by Ramakrishnan and Adelberg (1965a), whose analysis provided us with our present-day picture of the arrangement of the five structural genes for the isoleucine and valine biosynthetic enzymes. Their work demonstrated that, unlike the genes in the tryptophan cluster in E . coli and S. typhimurium, there was more than one repression recognition element and, therefore, presumably more than one operon or functional grouping.

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Very briefly, they presented evidence that the structural genes were arranged in the order B , C, A, D, and E corresponding to enzymes 2, 3, 1, 4, and 5 in the sequence (Ramakrishnan and Adelberg, 1965a). One class of valine-resistant mutant was that described biochemically several years earlier by Leavitt (Leavitt and Umbarger, 1962), in which the normally valine-sensitive enzyme (enzyme 2) was resistant. Such mutants, in which the ilv B gene was altered, were useful to locate the position of that gene on the chromosome since E . coli mutants lacking this enzyme have probably not yet been found, though probably mutations leading to abnormal enzyme have been observed (Ramakrishnan and Adelberg, 1965a,b). Another class of valine-resistant mutant was one in which the enzymes coded for by the ilv genes A , D , and E were derepressed (Ramakrishnan and Adelberg, 1965b). While the basis for valine-resistance in these mutants cannot yet be explained (Umbarger, 1969), they did provide a means of identifying the repression recognition element, designated ilv 0. Another class was found to have a derepressed ilv B gene, thus resulting in high levels of the valine-inhibited enzyme (Rarnakrishnan and Adelberg, 196513). Interestingly, these mutants had only the valine-sensitive enzyme derepressed indicating that the affected repression recognition region ilv P controlled only the adjacent ilv B gene. No mutations were found to affect regulation of the ilv C gene which is now known to be controlled in E . coli and S. typhimurium by substrate induction rather than by repression (Arfin et al., 1969). The first nonnatural analog of a branched-chain amino acid employed systematically as a biochemical and genetic probe was 5’,5’,5’-trifluoroleucine, a compound shown by Rennert and Anker (1963) to be incorporated into protein of E . coli and under suitable conditions, as yet not fully understood, to replace fully the leucine of proteins in that organism. This study, initiated by Calvo with S. typhimurium, led to the recognition of four classes of trifluoroleucine-resistant mutants. One very rare class contained an isopropyl malate synthetase (the end product-sensitive enzyme) that was resistant to inhibition by both leucine and trifluoroleucine (Calvo et al., 1969a). Another class was characterized as having normal leucine biosynthetic enzymes, but derepressed levels of all three (Calvo et al., 1969b). I n these, the lesion has affected the repression recognition site (designated leu 0) adjacent to the structural genes for the leucine biosynthetic enzymes. Another kind of trifluoroleucine-resistant mutant was one in which the lesion was not linked to the leu gene cluster. I n these, not only were the leucine biosynthetic enzymes derepressed, but also five isoleucine and valine biosynthetic enzymes. This property was of particular interest because it was known that for repression of the isoleucine and valine biosynthetic enzymes

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all three branched-chain amino acids had to be in excess (Freundlich

et al., 1962). The affected gene (leu S ) in these mutants was thus one that was required for generation of the “leucine excess” signal not only for interaction with the leu 0 region but with the presumed ilv 0 and ilv P regions as well. (Although ilv 0 and ilv P have not been demonstrated in S. typhimurium, the near-universal homology between gene order and function in E. coli and that in S. typhimurium serve to justify the presumption.) The leu S lesions have since been shown to result in leucyl-tRNA synthetases that have a reduced affinity for leucine. That difficulty in generation of the repression signal for multivalent repression of the ilv gene cluster accompanied the difficulty in activating leucine was not surprising in view of the findings of Neidhardt and his colleagues (1966) that under conditions of restricted valyl-tRNA synthesis the “excess valine” signal required for multivalent repression was not generated. That the “excess isoleucine” signal also required a normal activating enzyme was indicated by a study of E. coli thiaisoleucine resistant mutants (Szentirmai e t al., 1968). These mutants exhibited reduced levels of isoleucyl-tRNA synthetase. Subsequent genetic analysis of these has demonstrated that for the thiaisoleucine resistance and derepression observed in the original isolates, two mutational steps were required (Coker and Umbarger, 1970). One of these affected the structural gene, ilv S, of isoleucyl-tRNA synthetase, resulting in an enzyme with reduced activity and reduced affinity for isoleucine. The second gene, ilv U , appears to be a regulatory gene that leads to derepression of isoleucyl-tRNA synthetase when isoleucine is limiting. Cells containing only the ilv S lesion, although partially resistant to thiaisoleucine, appear to have a derepressed level of a poor synthetase. The derepression would appear to compensate at least in part for the reduced efficiency of the enzyme (resulting in about 50% of the wild-type activity) so that no depression of the isoleucine and valine biosynthetic enzymes results. When the ilv U mutation was introduced into an ilv S mutant, derepression of the poor synthetase was no longer possible, and only about 5% of the wildtype synthetase activity was observed in extracts. This introduction of ilv U was accompanied by an increase in thiaisoleucine resistance and derepression of the ilv gene cluster. [Actually in E. coli strain K-12, only the ilv ADE operon is derepressed when isoleucine is limiting, whereas in S. typhimurium all five enzymes are elevated (Dwyer and Umbarger, 1968) .] The introduction of ilv U into a wild-type organism led to levels of thiaisoleucine resistance and derepression less than those observed when ilv U and ilv S were both present. While an ilv U mutant exhibits

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a normal level of isoleucyl-tRNA synthetase when grown in minimal medium, it does not exhibit derepression of the synthetase when the isoleucine supply is restricted, as observed by Nass and Neidhardt (1967). It is of interest that the regulation of isoleucyl-tRNA synthetase is not multivalent but specifically responsive, via the ilv U gene, to isoleucine. I n some, but not all, thiaisoleucine-resistant mutants a third gene has been affected. This gene, ilv T, leads to a low level of derepression but not to thiaisoleucine resistance. It, too, may act synergistically with ilv S but its role is more obscure than that of ilv U . Thiaisoleucine has also been employed as a probe with S. typhimurium, but all the resistant mutants were those with an end product-insensitive threonine deaminase (Blatt, unpublished observations). This difference in pattern of analog resistance in two such similar organisms illustrates well the utility of examining the same analog in a variety of organisms that can be studied genetically. While the analog itself may not behave significantly differently in the two organisms, the permissible and feastble mutations leading to resistance in the two organisms may differ markedly. I n S. typhimurium, ilv S mutants were isolated as isoleucine auxotrophs with isoleucyl-tRNA synthetase with only 1/1000th the wild-type affinity for isoleucine (Blatt and Umbarger, 1970). In these organisms, the ilv S lesion itself led to derepression of all five isoleucine and valine biosynthetic enzymes. Another example of the advantage of utilizing a variety of analogs in a variety of organisms was noted when mutants comparable to those found by Calvo in S. typhimurium were sought in E . coli. However, trifluoroleucine was not a particularly effective inhibitor of E . coli, so that 4-azaleucine was employed an analog not particularly effective in S. typhimurium. Mutants of E . coli resistant to azaleucine were isolated that had, like leu S mutants of S. typhimurium, derepressed leucine biosynthetic enzymes and derepressed isoleucine and valine biosynthetic enzymes (except, of course, the inducible ilv C gene product that was not well induced because of the effectiveness of end product control on the valine-sensitive enzyme). However, this mutation was in an entirely different gene, and neither leucyl-tRNA synthetase, nor any leucine acceptor tRNA, were any different from those in wild-type E . coli. At the present time, the responsible gene, designated ad, is viewed as a necessary element along with normal leu 0, and leu S gene functions and excess leucine to generate the signal “excess leucine” and along with normal ilv 0, val S, ilv S, and ilv P gene functions and excess valine and isoleucine to generate the multivalent repression signal. Although one could formulate a model involving these elements it would

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seem premature to do so. Indeed, it might be anticipated that continued use of analog probes will reveal more elements with a role in regulation of these enzymes. From the results obtained with the branched-chain amino acids, one might doubt that the genetic analysis of the regulation of any amino acid biosynthetic pathway will be complete when evidence for a repression recognition region and a single unlinked regulatory element (as postulated by the Jacob-Monod model) has been found. The generation of repression signals may well be far more complex than we have anticipated and a combined genetic and biochemical probing may be almost essential. It is also quite possible that different analogs will result in different kinds of resistant mutants. Similarly, a comparative study of 8. typhimurium and E . coli with respect to the spectrum of regulatory elements that can be recognized may yield much information that will be complementary. This prediction is based upon the great extent of genetic homology that is found between the two species until one reaches the base-pair level of resolution. It is a t this latter level that the kind of effects that can be obtained by permissible changes in a base pair is determined. Such differences may account for the observation that a given analog may select for end product insensitive enzymes in one species and derepressed operons in another. VII. Outlook and Recommendations

The theme that I have tried to develop here, is that there is yet much to be learned by using metabolite analogs to select resistant mutants. The mutants so found may not only reveal biochemical relationships not earlier recognized, but also gene functions that are not recognized until altered. One might ask, cannot these interacting elements also be discovered by sufficiently detailed biochemical analysis? The answer is usually yes, but it does appear that some biological interactions are so tightly coupled that, without genetically controlled modification, resolution of the components involved would be difficult indeed. An example might be the proteins of the ribosomes that are tightly bound and then function as a single unit. An example a t another level might be the processes of transcription and translation of genetic information which, while apparently biochemically distinct, are so difficult to uncouple that it is not yet possible to find convincing evidence that control of gene expression is a t one level or the other. (Perhaps we should keep in mind that the goal in evolution was to achieve control of gene function and perhaps the means to achieve the end has not always been the same in each case.)

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While only a few examples have been chosen for discussion here, specifically analogs of the aromatic and branched-chain amino acids and histidine, it has been possible to show that their study has had considerable impact on the attempts to understand the mechanisms of regulation of gene expression in bacteria. The results have been quite enlightening to those of us who, following the introduction in 1961 of the very clear, concise and esthetically pleasing repressor-operator model, went off to our own biosynthetic and catabolic systems in bacteria, fungi, and even in developing embryos to invoke operators, repressors, and, more recently, promoters with complete abandon, only because we had no evidence to countraindicate them. This zeal on the part of the disciples was exactly opposite to the mode of operation used by Jacob and Monod (1961), who invoked only regulatory elements for which genetic and biochemical evidence was available. The more carefully examined picture currently seems quite complex. Indeed, the differences thus far revealed, even in such similar systems as these amino acid biosynthetic pathways, cause us to hesitate before concluding (paraphasing the Philistinism of a contemporary political revertant) that to have seen one operon is to have seen them all. One might wonder whether the regulation of gene expression, which must have evolved after the appearance of the function itself, might not in each case have developed along unique lines. While we might easily recognize analogous elements involved in repression and induction of certain gene functions, whether these elements are homologous in function and structure may require more detailed study. Metabolite analogs should continue to prove valuable in this area, which is one of the many that are extensions of what Demerec began at Cold Spring Harbor.

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