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Using Tryptophan Synthase to Prove Gene-Protein Colinearity: the Work of Charles Yanofsky

Open AccessPublished:November 18, 2005DOI:https://doi.org/10.1016/S0021-9258(19)37759-2
      The Enzymatic Conversion of Anthranilic Acid to Indole (Yanofsky, C. (1956) J. Biol. Chem. 223, 171–184)
      A Genetic and Biochemical Analysis of Second Site Reversion (Helinski, D. R., and Yanofsky, C. (1963) J. Biol. Chem. 238, 1043–1048)
      Studies on the Position of Six Amino Acid Substitutions in the Tryptophan Synthetase A Protein (Carlton, B. C., and Yanofsky, C. (1963) J. Biol. Chem. 238, 2390–2392)
      Amino Acid Substitutions in the A Proteins of Tryptophan Synthetase Mutants and Revertants (Carlton, B. C., and Yanofsky, C. (1965) J. Biol. Chem. 240, 690–693)
      The Amino Acid Sequence of the A Protein (α Subunit) of the Tryptophan Synthetase of Escherichia coli. V. Order of Tryptic Peptides and the Complete Amino Acid Sequence (Guest, J. R., Drapeau, G. R., Carlton, B. C., and Yanofsky, C. (1967) J. Biol. Chem. 242, 5442–5446)
      Charles Yanofsky was born in New York City in 1925 and was one of the early graduates of the Bronx High School of Science. In 1942, he entered the City College of New York to major in biochemistry, but his education was interrupted by his service in the Army during World War II, including active combat in the Battle of the Bulge. He eventually earned a bachelor's degree in 1948 and decided to go to graduate school to explore gene-enzyme relationships using Neurospora crassa. He went to Yale University, hoping to work with Edward Tatum. However, Tatum had just left Yale to return to Stanford University. Fortunately, Tatum's research associate, David Bonner, stayed on at Yale and took over the direction of Tatum's remaining group. Yanofsky joined Bonner's lab and earned his master's and doctoral degrees in microbiology in 1950 and 1951, respectively.
      Stimulated by the “one gene, one protein” hypothesis put forward by George Beadle and Edward Tatum, which was the subject of a previous Journal of Biological Chemistry (JBC) Classic (
      • Beadle G.W.
      JBC Classics
      ), Bonner's lab was focused on establishing the nature of the gene-enzyme relationship by examining Neurospora enzymes that appeared to be defective or missing in specific mutants. Yanofsky was involved in a project using niacin-requiring mutants to identify all the intermediates in the niacin pathway. The idea was to eventually exploit this knowledge in investigations on gene-enzyme relationships. Yanofsky was able to identify two intermediates accumulated by the mutants, quinolinic acid and a derivative of kynurenine. However, in his third and last year of graduate school, Yanofsky abandoned his niacin pathway studies and joined the rest of the lab in their attempt to prove the one gene, one protein hypothesis by demonstrating that all mutants altered at a single genetic locus lacked the specific enzyme that catalyzed the corresponding reaction. Having spent several years studying niacin and tryptophan metabolism in Neurospora, Yanofsky decided to investigate tryptophan synthase,
      This enzyme was initially named tryptophan desmolase, then tryptophan synthetase, and finally tryptophan synthase.
      1This enzyme was initially named tryptophan desmolase, then tryptophan synthetase, and finally tryptophan synthase.
      the only enzyme of the tryptophan pathway that had been identified experimentally. He was able to show that two tryptophan-requiring mutants lacked tryptophan synthase activity. Other members of the lab joined in, and soon they had isolated 20 additional mutants that were altered at the same locus—each lacked tryptophan synthase activity.
      In 1954, Yanofsky moved to Western Reserve University Medical School in Cleveland, Ohio to become an Assistant Professor of Microbiology. He decided to shift his research objectives to a well defined problem for which he could easily determine answers. He chose to focus on determining the missing reactions in the tryptophan biosynthetic pathway, which is the subject of the first JBC Classic reprinted here. At the time the paper was published, only two intermediates in the tryptophan pathway had been identified, anthranilate and indole. In this paper, Yanofsky uses a tryptophan auxotroph of Escherichia coli as a source for enzymes that convert anthranilic acid to indole. Using ammonium sulfate he was able to separate two fractions that catalyze successive reactions in the conversion of anthranilic acid to indole. One fraction catalyzed the formation of indole-3-glycerol phosphate from anthranilic acid and 5-phosphoribosyl-1-pyrophosphate, and the other fraction converted indole glycerol phosphate to indole and triose phosphate. With the aid of his graduate student Oliver Smith, Yanofsky was eventually able to identify several more intermediates in tryptophan synthesis including phosphoribosyl anthranilate and carboxyphenylamino-1-deoxyribulose 5-phosphate.
      In 1958 Yanofsky was persuaded to join the Biological Sciences Department at Stanford University. There, he decided to mount an all out effort to establish or disprove gene-protein colinearity. Continuing on with tryptophan synthase, Yanofsky and his postdoctoral fellow Irving Crawford established that the enzyme is composed of non-identical polypeptide chains. One subunit, TrpA, hydrolyzes indole glycerol phosphate to indole, and the second subunit, TrpB, covalently joins indole and l-serine to form l-tryptophan. This was the first demonstration that an enzyme could contain two dissimilar subunits. Yanofsky and Crawford also discovered that each subunit activates the other, suggesting that TrpA mutants could be isolated and their altered TrpA proteins assayed by measuring their ability to activate the TrpB subunit. Using this assay to purify TrpA mutant proteins, Yanofsky and his co-workers then identified the single amino acid changes in the mutant proteins and compared the positions of the amino acid changes with the order of the corresponding altered sites on a fine structure genetic map of the trpA gene. The second through fourth Classic reprinted here deal with these TrpA experiments.
      In the second Classic, Yanofsky determines the position of six amino acid substitutions in TrpA proteins derived from revertants of two trpA mutants. All six changes were found at the same amino acid position in the protein. This study was a prelude to establishing colinearity and specific features of the genetic code. The third Classic is a study in which the amino acid change in the TrpA protein of a second site revertant was determined. It was the initial demonstration that a second amino acid change in a protein could reverse the effects of a primary amino acid change. The details of the peptide isolation and sequence analyses with wild type and mutant TrpA proteins are presented in the fourth Classic. This paper once and for all established gene-protein colinearity in the trpA gene and TrpA protein of E. coli.
      Because thoroughness was part of his strategy, the Yanofsky group continued sequencing TrpA until its entire sequence had been determined. This was a huge accomplishment considering the procedures of protein chemistry available at that time. They published the detailed analysis that led to the determination of the complete amino acid sequence of TrpA in a series of five papers in one issue of the JBC (
      • Guest J.R.
      • Carlton B.C.
      • Yanofsky C.
      The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. I. Tryptic peptides.
      ,
      • Drapeau G.R.
      • Yanofsky C.
      The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. II. Amino acid sequences of two large tryptic peptides.
      ,
      • Carlton B.C.
      • Guest J.R.
      • Yanofsky C.
      The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. III. The chymotryptic peptides.
      ,
      • Drapeau G.R.
      • Yanofsky C.
      The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. IV. The cyanogen bromide fragments of the A protein.
      ). The final JBC Classic reprinted here is the final paper in the series, and it presents the complete amino acid sequence of the protein. There is one error in the sequence: Ile-36 was subsequently shown to be double Ile (Ile-36–Ile-37); thus the polypeptide has 268 rather than 267 amino acid residues.
      Yanofsky was appointed Herzstein Professor of Biology in 1967 and remains at Stanford today as an Emeritus Professor. The focus of his research continues to be the control of gene expression, in particular the molecular regulatory mechanisms of bacterial transcription. He has received numerous awards including the Albert Lasker Award in Basic Medical Research, the Genetics Society of America Medal, and the 2003 National Medal of Science. He was elected to the American Academy of Arts and Sciences in 1964 and the National Academy of Sciences in 1966. Yanofsky is past president of the American Society of Biological Chemists and of the Genetics Society of America and was a Career Investigator of the American Heart Association. In 1980, he and other Stanford scientists founded DNAX, a Palo Alto-based research institute now owned by Schering-Plough Corp. More detailed information on Yanofsky's research can be found in his JBC Reflections (
      • Yanofsky C.
      Using studies on tryptophan metabolism to answer basic biological questions.
      ).
      All biographical information on Charles Yanofsky was taken from Ref.
      • Yanofsky C.
      Using studies on tryptophan metabolism to answer basic biological questions.
      .
      2All biographical information on Charles Yanofsky was taken from Ref.
      • Yanofsky C.
      Using studies on tryptophan metabolism to answer basic biological questions.
      .

      References

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        • JBC Classics
        J. Biol. Chem. 1944; 156 (http://www.jbc.org/cgi/content/full/280/12/e9): 683-690
        • Guest J.R.
        • Carlton B.C.
        • Yanofsky C.
        The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. I. Tryptic peptides.
        J. Biol. Chem. 1967; 242: 5397-5412
        • Drapeau G.R.
        • Yanofsky C.
        The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. II. Amino acid sequences of two large tryptic peptides.
        J. Biol. Chem. 1967; 242: 5413-5421
        • Carlton B.C.
        • Guest J.R.
        • Yanofsky C.
        The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. III. The chymotryptic peptides.
        J. Biol. Chem. 1967; 242: 5422-5433
        • Drapeau G.R.
        • Yanofsky C.
        The amino acid sequence of the A protein (α subunit) of the tryptophan synthetase of Escherichia coli. IV. The cyanogen bromide fragments of the A protein.
        J. Biol. Chem. 1967; 242: 5434-5441
        • Yanofsky C.
        Using studies on tryptophan metabolism to answer basic biological questions.
        J. Biol. Chem. 2003; 278: 10859-10878