Mutations tryptophan synthetase

There is currently some uncertainty about the complexity of the tryptophan gene-enzyme relationships in B. subtilis. Whitt and Carlton [46,47] have noted pleiotropic effects. Most recently [46] they have found that the pleiotropy is limited to elimination of InGPS activity by mutations in either the trpD or trpF genes, which are primarily concerned with PRT and PRAI activity, respectively. The pleiotropic effects are shown in Fig. 3 by dotted lines and may indicate that the enzymes function as aggregates in vivo or may represent effects on translation similar to polarity effects. Hoch et al. [48], on the other hand, have not found these pleiotropic effects and report essentially one gene-one enzyme activity [when the individual activities of the tryptophan synthetase a and p2 subunits (Fig. 1) are included]. The nonconformity in the results of different investigators may be due to the use of different mutants and to different methods of preparation, affecting enzyme stabilities. 

Fig. 4. Diagram of the tryptophan operon plus two neighboring genes. The gene designations for both E. coli and S. typhimurium are given. The supX locus has not been identified in E. coli. Controlling elements PI, the promoter associated with the operator 0, the operator P2, the low-efficiency internal promoter. Enzymes AS-I, anthianilate synthetase component I PRT, phosphoribosyl transferase (AS-II, anthianilate synthetase component II) PRAI, phosphoribosyl anthianilate isomerase InGPS, indoleglycerol phosphate synthetase TS-a, tryptophan synthetase a-chain TS-)S, tryptophan synthetase j8-chain. RUM, region of unusual mutations in S. typhimurium.

Studies on bacterial mutation have shown that the induction of a specific enzyme is usually accompanied by an increase in activity of several other enzymes involved in the same catabolic or anabolic pathways. When E. coli are grown in the presence of galactosides, the formation of j8-galactosidase is induced, and it has been established that the new enzyme formed (1000 times the activity present in the wild type of E. coli) is not derived from the activation of preexisting proenzyme, but from the net synthesis of new enzyme. In 1953, Monod [213] discovered that the activity of tryptophan synthetase is inhibited by tryptophan and certain of its analogs. It was established that the inhibition or repression resulted from a block of the enzyme biosynthesis. 

With these studies I was committed to working with tryptophan synthetase and I moved on eagerly to further analyses of the enzyme changes resulting from mutations. By this time the techniques of mutant isolation in Neurospora had improved appreciably so that Bonner and I were able to obtain a large set of non-identical tryptophan synthetase mutants. 

I was surprised to find that the growth in the control flask was in fact due to a suppressor mutation at a locus unlinked to the tryptophan synthetase locus. To my satisfaction, extracts of the suppressed mutant contained low but detectable levels of tryptophan synthetase activity. Further analyses showed that the restored enzyme was indistinguishable from the wild type enzyme. Encouraged by these results I put other studies aside and, with the aid of Miriam Bonner and my wife, Carol, examined many tryptophan synthetase mutants for reversion by unlinked suppressor mutations. Our hopes were realized for we readily detected several suppressor mutations. These, for the most part, were allele specific, i.e. each suppressor gene suppressed only one or two of the set of tryptophan synthetase mutants tested. In addition, the suppressed mutants invariably contained tryptophan synthetase activity. ... 

During this period I was frustrated by my inability to obtain tryptophan synthetase in pure form from Neurospora. Therefore, I searched for a more suitable enzyme for studies on protein structure alterations resulting from mutations. I characterized d- and L-serine deaminases from Neurospora - with this objective in mind but was unable to develop a convenient isolation procedure for mutants lacking these enzymes and so stopped these investigations. 

Yanofsky, C. and Crawford, I. P. (1959) The elfects of deletions, point mutations, reversions and suppressor mutations on the two components of the tryptophan synthetase of Escherichia coli. Proc. Nat. Acad. Sci. 45,1016-1026. 

Yanofsky, C., Helinski, D. and Malino, B. (1961) The effects of mutations on the composition and properties of the A protein of Escherichia coli. tryptophan synthetase. Cold Spring Harb. Symp. Quant. Biol. 26, 11-23. 

Yanofsky, C. and Horn, V. (1972) Tryptophan synthetase o-chain positions affected by mutations near the end of the genetic map of trpA of Escherichia coli. J. Biol. Chem. 247, 4494-4498. 

Mutant A mutations with decreased, but detectable, tryptophan-tRNA synthetase activity... 

There have not been any reports of strains resistant to tryptophan analogs as a result of mutant tryptophanyl-tRNA synthetase. However, three laboratories have independently isolated strains of E. coli with partial tryptophan auxotrophies (bradytrophs) as a result of mutations in what appears to be the structural gene, trpS, for tryptophanyl-tRNA synthetase [187,188,214,215]. The partial auxotrophy was overcome by either tryptophan or indole supplementation but not by anthranilate. 

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