REFLECTIONS
Using Studies on Tryptophan Metabolism to Answer Basic
Biological Questions
Charles
Yanofsky
From the Department of Biological Sciences, Stanford University,
Stanford, California 94305
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INTRODUCTION |
In my youth I was overwhelmed by the variety of forms of life
around me. Yes, while growing up in New York City! As a student at the
Bronx High School of Science my teachers made every effort to convince
me that no pursuit could be more exciting or rewarding than searching
for explanations for the basic processes common to life. I agreed, but
I knew this decision was insufficient, for I would have to choose the
area of science that was just right for me. I was aware that major
unanswered questions existed in all fields of science, particularly
regarding the relationship of biochemistry to genetics, the two
subjects that interested me most as a high school student. I decided to
major in biochemistry, and enrolled at the City College of New York. I
completed a year and a half of college study before being drafted into
the army in the spring of 1944. I served in the infantry as a cannoneer during World War II. I fought in the Ardennes in the Battle of the
Bulge. Understandably this was an awesome experience. Upon returning to
college after the war I was more determined than ever to pursue a
career in research. When faced with selecting a Ph.D. program to apply
to, I received excellent advice from a knowledgeable professor and
textbook author, Benjamin Harrow, chairman of the Biochemistry
Department at City College of New York. He suggested exploring
gene-enzyme relationships with Neurospora crassa as the
ideal project for me. I agreed and applied to do my graduate work with
George Beadle at Caltech or Edward Tatum at Yale. I was rejected by
Caltech but fortunately was accepted by Yale.
As it turned out, my mentor in graduate school at Yale was not Edward
Tatum; it was David Bonner. Bonner had moved with Tatum from Stanford
to Yale and had become his research associate. During the year I
applied for admission to Yale, Tatum decided to return to Stanford.
Fortunately for me, Bonner stayed on at Yale and took over direction of
Tatum's remaining group. Bonner, a wonderful advisor, believed it was
in the best interests of both student and advisor to have each student
work independently on a well defined project. If successful, he said,
we would receive partial credit for our discoveries and would qualify
for a faculty position. For most beginning graduate students, selecting
a project and deciding how to proceed is relegated to your research
mentor and would reflect his or her research preferences. By choosing a
specific scientist as your advisor you recognize the importance of his or her contributions. In my initial meeting with Bonner at Yale in June
of 1948, as I recall, he handed me a fuzzy culture of a
niacin-requiring mutant of Neurospora and gave me advice on how to go about identifying the niacin pathway intermediate this mutant
was presumed to accumulate. Our ultimate goal, he said, was identifying
all the intermediates in the niacin pathway so this knowledge could be
exploited in investigations on gene-enzyme relationships. I was the
only laboratory member assigned this type of project, probably because
Bonner was aware that my background was principally in biochemistry.
This project captured my full attention, and fortunately, I was
successful. We identified two intermediates accumulated by
niacin-requiring mutants, quinolinic acid and a derivative of
kynurenine. The knowledge I acquired in these studies served as a
valuable resource in decision making throughout the early stages of my career.
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Reflections: Questions, Answers, and More Questions |
Upon reviewing my research accomplishments and considering
what I might emphasize in this article, I was most impressed by the
variety of basic biological questions the members of my group have
addressed. Early in my career I decided that one of my primary research
objectives would be to provide a thorough understanding of all aspects
of tryptophan metabolism and to use this knowledge in explaining basic
processes of biology. In fact, tryptophan metabolism was the focus of
most of my research. However, during the early stages of my career I
did not appreciate the variety of scientific questions that I would
have the opportunity to address using tryptophan metabolism as my
experimental system. Our studies contributed to knowledge on the niacin
and tryptophan biosynthetic pathways, enzyme structure/function
relationships, organization of genes and operons, the existence of
gene-protein colinearity, the molecular basis of suppression, coupling
of transcription with translation, regulation of transcription, how
tryptophan and tryptophan-tRNA serve as regulatory signals, and the
regulatory mechanisms microorganisms use to control tryptophan
synthesis and its degradation. The unanticipated role of RNA in
regulation, transcription attenuation, was and continues to be one of
our major interests. We had no inkling until the 1990s, when bacterial genomes were beginning to be sequenced, that attenuation was so widely
used in nature. While we were conducting our investigations on
tryptophan metabolism evolutionary questions continually arose. As soon
as we understood the features of tryptophan metabolism in one organism
we wished to know whether other organisms use the same genes,
reactions, and regulatory processes. Despite my personal commitment to
tryptophan metabolism, in the early 1980s I returned to studies with
N. crassa as an experimental organism, addressing other
important questions. The lesson to be learned from my experiences, I
believe, is to always be on the alert. Important unanswered questions
you never anticipated will invariably arise from the results of your
current research. It may develop that your chosen experimental system
is ideal for answering these questions. Throughout this article I will
describe examples taken from my career, where answers led to questions
I felt we should address.
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The One Gene-One Enzyme Relationship |
When I arrived at Yale 1n 1948 most members of the Bonner
group were coping with the most significant question then concerning the Neurospora scientific community: how to establish the
nature of the gene-enzyme relationship. It was some years after Beadle and Tatum (1) had first proposed the one gene, one enzyme, one
biochemical reaction hypothesis. Following the pioneering studies of
Garrod in the early 1900s, linking heredity with metabolism, there were
numerous observations relating metabolic defects with genetic
disorders. Beadle and Tatum cemented this relationship in the early
1940s by selecting an organism, N. crassa, that could be
used to isolate nutritional mutants. These mutants could then be
genetically characterized to establish whether their inability to carry
out specific biochemical reactions was because of mutations in specific
genes. Most importantly, they observed that there was a one to one
relationship between gene and biochemical reaction. Despite these
findings, when I was completing my graduate studies in 1951 most
scientists were skeptical of the validity of the one gene-one enzyme
concept. At this time very little was known about the molecular nature
and structure of genetic material or the structure of proteins, and
virtually nothing was known about protein synthesis. It was not until
the early 1950s that the findings of Hershey and Chase (2) and an
earlier finding by Avery et al. (3) convinced most of us
that genetic material was most likely DNA, and it was not until 1953 that Jim Watson and Francis Crick (4) described their elegant structure
for DNA. Following these major contributions we accepted as proven that
the genetic material of most organisms was double-stranded DNA.
Furthermore, it was not until the late 1950s that Seymour Benzer's (5)
fine structure genetic analyses with the rII locus of phage T4 equated the genetic map with the structure of DNA. Similarly, it was not until
the early 1950s that Sanger's studies (6) with insulin established
that proteins consist of linear sequences of amino acids.
While I was in graduate school the goal considered most important by
members of the Beadle-Tatum school was to identify a specific enzymatic
reaction for which defective mutants could be isolated and then
determine whether these mutants lacked that enzymatic activity. Our
hope was that studies like these would provide definitive proof for the
one gene-one enzyme hypothesis. Several members of the Bonner group
were following this approach. Naomi Franklin, Otto Landman, Gabriel
Lester, and Howard Rickenberg were examining one of the most popular
experimental enzymes during this period, 
-galactosidase, from both
Neurospora and Escherichia coli. They were hoping
to use the knowledge and techniques being provided by Monod, and
subsequently by Jacob and Monod and their exceptional coworkers, to
explore the Beadle-Tatum gene-enzyme concept more directly. Impressed
by this overriding goal of my mentor and the determination of my fellow
students, I decided that I too should follow this path. In my third and
last year of graduate study, 1950-1951, I abandoned my niacin pathway
studies and initiated a search for the ideal "gene-enzyme"
experimental system.
No one in our group at Yale was contemplating what today would be
considered the most obvious experimental approach: isolating and
sequencing a specific gene and comparing this sequence with the amino
acid sequence of its polypeptide product. Neither genes nor proteins
could be analyzed in this way; we did not yet know that genetic
material was DNA or that proteins consisted of linear sequences of
amino acids. At this time the prevailing view in the field of genetics
was that chromosomes consist of linear arrays of genes arranged like
"beads on a string." It was assumed that each gene was indivisible
by genetic recombination. If these views were correct how could we
determine the relative positions of independent mutational changes in a
specific gene, except by structural analysis, which was not possible?
We decided that our next step on the gene-enzyme problem should be to
demonstrate convincingly that all mutants altered at a single genetic
locus lack the specific enzyme that catalyzes the corresponding reaction.
By the late 1940s numerous nutritional mutants of N. crassa
had been isolated, many requiring the same metabolite. It was evident
that amino acids, vitamins, purines, and pyrimidines are all
synthesized by sequential enzyme-catalyzed reactions, mostly in
separate pathways. However, these pathways were just beginning to be
defined. Genetic analyses with these mutants established a very
impressive one-to-one relationship between altered gene and loss of a
specific biochemical reaction; this was the experimental basis of the
Beadle/Tatum concept. It was also evident that a unique set of genes
was associated with each metabolic pathway. However, very few of the
enzymes in each newly discovered pathway had been identified, and those
that were known did not catalyze reactions that were defective in the
nutritional mutants that had been isolated. One of the earliest
opportunities to examine mutants lacking a specific enzyme was provided
by the findings of Umbreit et al. in 1946 (7). They
demonstrated that extracts of wild type Neurospora contain
an enzyme they named tryptophan desmolase, which catalyzes the last
reaction in tryptophan synthesis, the covalent joining of indole with
L-serine to form L-tryptophan. Tryptophan-requiring mutants of Neurospora had been
identified that could not grow on indole; therefore these mutants
should lack this enzyme activity if the Beadle/Tatum hypothesis were correct. Joseph Lein and Dave Hogness, of Hershell Mitchell's laboratory at Caltech, examined extracts of one such mutant, named td1, and reported that yes, it did lack tryptophan
desmolase activity (8, 9).
Having spent my first 2 years studying niacin and tryptophan metabolism
in Neurospora, I decided that tryptophan desmolase was
promising as a potential subject for gene-enzyme analyses. I initiated
my studies by partially purifying and further characterizing the wild
type enzyme and confirming the absence of tryptophan desmolase activity
in extracts of mutant td1. I also examined a second mutant
altered at the same locus, mutant td2, and showed that it
too lacked tryptophan desmolase activity (10). Excited by the
simplicity of this enzyme assay and these positive results, members of
the Bonner group turned to isolating 20 additional mutants defective in
the conversion of indole to tryptophan. We showed that each was
genetically altered at the td locus and each lacked
tryptophan desmolase activity. These initial findings were very
encouraging, and they supported the basic assumption of the Beadle/Tatum concept.
In the course of my studies with mutant td2 one culture grew
in media lacking tryptophan. Instead of discarding this culture, we
analyzed it genetically and discovered that its ability to grow without
tryptophan was due to an unlinked suppressor mutation. The properties
of this suppressed td mutant raised a new, then unanswerable, question. How does a suppressor mutation, a
mutation in a gene other than the td gene, restore growth without
tryptophan? My enzyme analyses revealed that the suppressor
mutation acted by restoring the organism's ability to form an active
tryptophan desmolase (10). Probing still further, I observed that the
td2 suppressor gene was allele-specific; it had no effect on
mutant td1. Obviously, then, mutants td1 and
td2 must have different alterations at the td
locus. We next performed "reversion" analyses with all our
td mutants and isolated several additional suppressors. Most
of these restored tryptophan desmolase activity only when combined with
their respective td mutant allele. On the basis of these
findings we rephrased our previous question, as follows. If there
is a one-to-one relationship between gene and enzyme and only td
mutants lack tryptophan desmolase activity, how does a mutation in a
gene distinct from the td locus restore this enzyme activity? My
thoughts on possible explanations temporarily diverted attention from
my primary objective, establishing the basis of the one gene-one enzyme
relationship. I considered our suppression findings to be extremely
interesting and believed that their explanation might provide
additional insight into this relationship. This experience, I believe,
was largely responsible for many of my subsequent decisions on how to
proceed in planning future research. I decided then that our knowledge
of basic biological processes was so poor it would be foolish to ignore
interesting unexplained observations. Following this line of reasoning
I set out to compare the properties of tryptophan desmolase isolated
from the wild type strain and from several suppressed mutants.
Throughout this period we were frustrated at how little we could do
experimentally. The existing molecular technology was clearly
inadequate. With a close friend and former member of the Bonner group,
Sigmund Suskind, then a postdoctoral fellow performing immunological
research at another institution, we designed a different approach that
we thought might provide additional insight into the gene-enzyme
relationship, The question we set out to answer was the following.
Does suppressible mutant td2, but not non-suppressible mutant
td1, produce an inactive form of the tryptophan desmolase enzyme?
Using my partially purified wild type enzyme as antigen, Suskind
prepared a rabbit antiserum that inhibited wild type tryptophan desmolase activity. We used this antiserum in a successful weekend experiment at Yale, analyzing extracts of mutants td1 and
td2 for an inactive tryptophan desmolase-like protein that
would cross-react with our antiserum (11). Mutant td2
extracts did in fact contain such a cross-reacting material, for which
we coined the term "CRM," whereas extracts of mutant td1
did not. Comparable analyses were then performed with extracts of our
other td mutants. All our suppressible mutants were shown to
be CRM+, whereas all our non-suppressible mutants were
CRM
. These findings implied, incorrectly, that
suppression can restore a functional enzyme only if a mutant produces
an inactive form of the wild type enzyme. (There are several reasonable
explanations for our inability to isolate suppressors of our
CRM Neurospora mutants, which probably had
chain termination mutations in the td gene.)
On the basis of our findings I drew a number of interesting
conclusions. I presented these at a very exciting symposium entitled "Enzymes, Units of Biological Structure and Function" held at the
Henry Ford Hospital in Detroit in 1955 (Fig.
1) (12). My interpretations were of
course influenced by new knowledge on DNA and protein structure and the
mechanism of protein synthesis. I concluded that "the td
locus is the only chromosomal area which directly controls tryptophan
synthetase formation" (the accepted name had just been changed from
desmolase to synthetase). I also concluded that "the td
locus represents a physiologically indivisible unit, damage to any part
of which results in a defect in tryptophan synthetase formation." I
stated that "it would seem likely that different portions of the
td locus are concerned with the synthesis of different parts
of the tryptophan synthetase molecule." In attempting to explain how
a suppressor mutation restores enzyme activity I postulated that
"some product of a suppressor gene cooperates with the altered
template in the formation of small amounts of tryptophan synthetase."
Looking back on these interpretations, they were all naive guesses, but
they proved to be correct. Unfortunately the experimental tools and
approaches needed to establish their molecular validity were not
available. These studies on missense suppression preceded the enormous
interest in suppression aroused by studies on the genetic code and on
nonsense mutations. As is so often the case, the significance of a
finding is not appreciated until additional relevant knowledge is
acquired.
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Fig. 1.
Early speculation on how a suppressor
mutation might restore synthesis of an active tryptophan synthetase
protein (TS) in mutant td2 but not
mutant td1. It was assumed that the td
gene was altered differently in the two mutants. This allowed the
product of a specific suppressor gene to act on the altered template of
mutant td2, the CRM+ mutant, to produce an active tryptophan
synthetase enzyme. Copied with permission from Academic Press
(12).
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Changing my Experimental Organism |
At this stage in my career I was deeply committed to doing
everything I could to provide additional insight into the gene-enzyme relationship. I was disappointed at the difficulty I was experiencing attempting to purify the tryptophan synthetase of Neurospora
and initiated a search for a more suitable experimental enzyme. My first thought was to identify an enzyme in the tryptophan to niacin pathway from E. coli or Bacillus
subtilis, because these organisms were developing as more
ideal experimental subjects for biochemical analyses. I performed
radioisotope-labeling experiments with these two organisms, hoping to
show that one or both synthesizes niacin from tryptophan. My
findings provided a disappointing conclusion; neither organism
synthesizes niacin from tryptophan (13). This negative result
eliminated enzymes of the niacin pathway from my list of possibilities.
While performing these studies I was offered a faculty position in the
outstanding Microbiology Department at the Western Reserve University
School of Medicine. I decided to accept their offer and left Yale for
Cleveland in 1954. As a beginning Assistant Professor I felt it would
be wiser to shift my research objectives to a well defined problem, one
for which I could foresee obtaining definitive answers. I relied on my
prior scientific experience and chose determining the missing reactions
in the tryptophan biosynthetic pathway. Although many different classes
of tryptophan auxotrophs had been isolated in Neurospora,
E. coli, and other organisms, only two intermediates in the
tryptophan pathway had been identified, anthranilate and indole. I
chose an enzymological approach in attempting to identify the
intermediates in the pathway and initiated my studies by analyzing
extracts of wild type and different classes of tryptophan auxotrophs of
E. coli.
My efforts focused on unidentified intermediates in the tryptophan
biosynthetic pathway were successful. Using an enzymological approach
we succeeded where others who had employed in vivo
approaches had failed. The principal reason for this is that the
unidentified intermediates in the tryptophan pathway are all
phosphorylated. Phosphorylated intermediates accumulated in
vivo would have been dephosphorylated and therefore inactive when
fed to a mutant. With the aid of my graduate student Oliver Smith, the
following intermediates were identified: phosphoribosyl anthranilate,
carboxyphenylamino-1-deoxyribulose 5-phosphate, and indole-3-glycerol
phosphate (IGP). The initial precursor of the tryptophan pathway,
chorismic acid, was isolated and identified by Frank Gibson, working
with his own group in Australia. Chorismate also serves as precursor of
the other aromatic amino acids. With the identification of these
additional compounds, the precursor and all the intermediates in the
tryptophan biosynthetic pathway were known.
While conducting these studies I made an unanticipated observation that
subsequently proved to be of enormous benefit in our colinearity
studies. I observed that many tryptophan auxotrophs of E. coli, when cultured on growth-limiting levels of tryptophan, produced 20-50 times more tryptophan synthetase than the wild type
strain. I thought that the day might come, as it did, when I could
exploit this observation to overproduce mutant proteins for
purification and analysis. I was aware of the regulatory significance of this observation and concluded that ultimately we should address the
regulatory mechanism(s) responsible for this increase.
Despite this temporary diversion in the mid-1950s, I was still
committed to establishing the nature of the gene-enzyme relationship. Knowledge about genes, proteins, and protein synthesis was improving, so much so that the gene-enzyme relationship was redefined. The question had matured to the following. Is the nucleotide sequence of a gene colinear with the amino acid sequence of the corresponding protein? During this period we learned many new facts about
tryptophan synthetase. I thought it might prove to be an ideal enzyme
for addressing the colinearity question. Our continuing investigations with this enzyme, from both Neurospora and E. coli, suggested that it may catalyze the last two reactions in
tryptophan formation, the cleavage of IGP to indole and the coupling of
indole with serine to form tryptophan. However, there were two
observations we could not explain: free indole could not be detected as
an intermediate in the conversion of IGP to tryptophan, and the rate of
conversion of IGP to indole was lower than its rate of conversion to
tryptophan (14). We then had to ask the following question. Does
the enzyme catalyze a third reaction in which IGP and serine react with
one another to form tryptophan, or is indole truly the intermediate,
and it remains within the enzyme complex? This puzzle was
not satisfactorily solved until the late 1980s. Then, the elegant
structural solution for the 
22 tryptophan
synthase (name changed again) enzyme complex of Salmonella
by Hyde et al. (15) revealed that there is a physical tunnel
in this enzyme complex connecting the active site of one polypeptide
subunit, , where indole is produced from IGP, to an active site of
the second subunit, 2, where indole reacts with
L-serine to form L-tryptophan (15, 16). As you
might imagine, it was comforting to have our confusing early
observations explained unambiguously by structural and enzymatic studies.
At this stage in my career everything was going well for me at Western
Reserve Medical School. I had quality co-workers and I thoroughly
enjoyed my interactions with my fellow faculty members, Howard Gest,
John Spizizen, David Novelli, Bob Greenberg, and Abe Stavitsky.
However, in 1957 I was contacted by Victor Twitty, chairman of the
Department of Biological Sciences at Stanford University, and offered a
faculty position. Despite my initial disinterest in considering this
appointment, I accepted their offer for a variety of reasons, including
my learning that Arthur Kornberg's department would be moving to the
Stanford campus (to the Stanford Medical School, which was being
relocated from San Francisco). Of historical interest, when I arrived
at Stanford the laboratory space I was provided was in the basement of
old Jordan Hall and was the space previously occupied by Ed Tatum and
his research team. I truly was treading in Tatum's footsteps!
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Proving or Disproving Gene-Enzyme Colinearity |
When setting up my laboratory at Stanford in January of 1958, I decided that the time had come to mount an all out effort to
establish or disprove gene-protein colinearity. I was joined in this
project by an outstanding young postdoctoral fellow, Irving Crawford,
who was recommended to me by Arthur Kornberg. In his exploratory
studies with tryptophan synthetase from E. coli, Irving was
first to establish that the enzyme is a complex composed of non-identical polypeptide chains. One subunit, TrpA (TSase ), hydrolyzes IGP to indole, whereas the second subunit, TrpB (TSase 2), covalently joins indole and L-serine to form
L-tryptophan (17). However, the enzyme from
Neurospora is a single polypeptide chain. In what proved to
be an extremely valuable observation for our subsequent colinearity
studies, Irving found that each E. coli subunit activates
the other subunit in the reaction that subunit performs alone. This
finding suggested that we might be able to detect and assay each
inactive TrpA mutant protein enzymatically by measuring its ability to
activate the TrpB subunit in the indole plus serine to tryptophan
reaction. This expectation proved to be correct; we routinely assayed
each mutant TrpA protein during its purification by measuring its
activation of TrpB.
We next prepared a set of pure mutant TrpA proteins, each presumably
with a single inactivating amino acid change. Good fortune helped us
again, for in 1958 Vernon Ingram described an elegant method,
"peptide fingerprinting," which he had used to detect peptides with
single amino acid changes in mutant human hemoglobins (18). This
approach seemed ideal for what we wished to do. If we could identify
the single amino acid change in each of our mutant proteins we would
then only have to compare the positions of these amino acid changes in
TrpA with the order of the corresponding altered sites on a fine
structure genetic map of the trpA gene to prove or disprove
gene-protein colinearity. I knew that we could construct a fine
structure genetic map of trpA using phage P1, based on a
previous genetic study I performed with Ed Lennox (19). I was confident
that very shortly we would convincingly prove or disprove gene-protein colinearity.
As one's research accomplishments become better known to the
scientific community, increasing numbers of young scientists will apply
to join your group. This necessitates making decisions on what size
group you consider optimal and how many projects you wish to attack.
Because I enjoyed working at the bench, I felt that I would have
sufficient time to serve as advisor to a maximum of about four graduate
students and four to six postdoctoral fellows. I had decided sometime
earlier to employ two research assistants who would work closely with
me, one to perform genetic analyses and the second to carry out
biochemical procedures. I was extremely fortunate that an exceptionally
bright and competent assistant, Ginny Horn, joined my group in 1958. She performed many of our genetic analyses for over 40 years. A series
of talented assistants provided my biochemical "hands."
In the early 1960s the colinearity problem was well publicized.
Progress was being made in several laboratories, and it was discussed
at many scientific meetings. Outstanding young scientists who joined my
group to work on this problem were Don Helinski, Ulf Henning, and
Barbara Maling, followed by Bruce Carlton, John Guest, and Gabriel
Drapeau. Of considerable aid in our genetic analyses was the use of
overlapping trpA deletion mutants for initial localization
of primary mutations on our fine structure genetic map of the
trpA gene. Thus we exploited the approach used so
successfully by Seymour Benzer. We obtained trpA deletion
mutants by selecting bacteria resistant to phage T1 and screened for
those requiring tryptophan for growth. These arose because the
tonB locus is close to the trpA locus, and
tonB deletions that confer resistance to phage T1 often
extend into trpA. The contributions of the individuals
mentioned above and those who replaced them established colinearity of
the TrpA protein with the trpA gene in the early 1960s. I
first described our findings supporting colinearity at the Cold Spring
Harbor Symposium of 1963 (20). Our complete proof was published in 1964 (21) (Fig. 2). Because thoroughness was
an essential element of my strategy, we continued our protein
sequencing analyses until the entire amino acid sequence of the
268-residue TrpA protein was completed in 1967 (22). This was by no
means a trivial feat, given the technology then available. At that time
I believe the TrpA protein was the longest polypeptide to have been
completely sequenced.
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Fig. 2.
Colinearity of the trpA gene
of E. coli with the tryptophan synthase
chain. The genetic map of trpA
(double line above) reflects the
relative positions of the mutationally altered sites examined in the
trpA gene. This map is based on recombination frequencies
observed in mutant by mutant crosses. The corresponding polypeptide
chain is shown below with the numbered position of each amino acid
change (and the amino acid change itself) indicated for each mutant.
Note that two ochre nonsense changes define the ends of the
genetic map. Copied with permission from the Annual
Review of Biochemistry (101).
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As mentioned, we were not the only group addressing the colinearity
problem. Comparable studies were being performed with the alkaline
phosphatase of E. coli by Rothman, Garen, and Levinthal, the
lysozyme of phage T4 by Streisinger and Dreyer, the rII locus of phage
T4 by Benzer and co-workers, and by others working with different
gene-protein systems (23). During this period Sydney Brenner and his
co-workers also established gene-protein colinearity using a simpler,
ingenious strategy (24). They reasoned that the length of a polypeptide
chain should be determined by the location of the first in phase stop
codon in a coding region. Applying this logic they mapped nonsense
mutations to different positions in the head protein gene of phage T4
and demonstrated that the length of the head protein fragments these
mutants produced correlated with the locations of the stop codon
mutations on the genetic map of the head protein gene.
Despite the many findings in the 1960s supporting gene-protein
colinearity, we of course were unaware at the time of the existence of
splicing, differential splicing, and trans-slicing, common processes
that would have weakened our confidence in our conclusion.
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Turning to the Genetic Code |
Technology did not exist in the 1960s that would allow us to
determine the nucleotide changes in our mutated genes. Fine structure genetic mapping, a la Benzer, was the only effective strategy to
characterize a mutated gene. However, much was being learned about
mutagenesis and mutagen specificity, primarily, as I recall, from
studies by Seymour Benzer and Ernst Freese. One of their objectives was
to use mutagens with differing specificities to help in deciphering the
genetic code. If the code is a triplet code, as deduced by Crick and
co-workers (25), and if chemical mutagens do induce specific nucleotide
changes in DNA, then it should be possible to correlate specific amino
acid changes in any protein with presumed induced nucleotide changes in
the specifying gene. This indirect approach, if applied to all 20 amino
acids, should reveal the nature of the genetic code. We felt that we could use it with our system to solve the genetic code. This basic question was as follows. Can we deduce the genetic code by
analyzing the amino acid changes in the TrpA proteins of trpA mutants
and their revertants, produced with mutagens with differing known specificities? The following members of my group adopted this strategy: John Guest, Manny Murgola, Hillard Berger, and Bill Brammer.
They successfully used specific mutagens to produce multiple classes of
revertants from each of our trpA mutants and identified the
amino acid changes in many mutant and revertant proteins. This approach
also laid the groundwork for impressive subsequent studies on
mechanisms of suppression, carried out by Manny Murgola. While these
studies were under way the entire scientific world, us included, was
startled to learn that Marshall Nirenberg had developed an elegant
in vitro method that would allow the complete genetic code
to be deciphered quickly and unambiguously. Despite our inability to
compete with Nirenberg, we did obtain appreciable in vivo
data supporting his deductions for over 45 codons (26). We also
performed mutant by mutant crosses with mutants bearing different amino
acid changes at the same TrpA position and showed that genetic
recombination can occur within a coding triplet and yield a recombinant
amino acid (27).
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Other Gene-Protein Issues |
While performing studies on the proteins of "revertants"
of trpA mutants, Don Helinski noticed that some presumed
revertants retained the original mutant amino acid change. Prototrophy
in these revertants was because of a compensating, second amino acid change. We named this phenomenon "second site reversion" (28). Helinski also observed that the second site amino acid change in one of
these "revertants," when introduced alone in TrpA, also inactivated
the protein. Thus, two inactivating single amino acid changes, when
combined in the same protein, could restore enzyme activity. These
findings could not be explained at the time, and it was apparent that
they would have to await structural examination. When three-dimensional
structure of the tryptophan synthase enzyme complex of
Salmonella was solved in the late 1980s it was observed that
the residues altered in the second site mutants were all in close
spatial proximity in the active site of the TrpA subunit (15). Computer
graphics modeling predicted that the compensating residue changes acted
by restoring the proper geometry of the substrate binding site in TrpA
(29).
| |
Returning to Suppression |
A familiar question resurfaced in the early 1960s in our
studies with trpA mutants. How does a mutation in a
specific suppressor gene permit a trpA missense mutant to produce a
functional enzyme? Stu Brody purified the active TrpA protein of
one suppressed missense mutant and used peptide fingerprinting analyses
to show that the active protein has the wild type residue, Gly, rather
than the mutant residue, Arg, at the critical position in the TrpA
protein (30). He postulated that suppression causes translational
misreading of the mutant Arg codon, leading to the insertion of the
wild type amino acid, Gly, at the critical position in the protein. When Brody became aware of the role of transfer RNAs in protein synthesis, he postulated that his missense suppressors, like previously characterized nonsense suppressors, might produce an altered transfer RNA that incorporates the wrong amino acid. This proposal was confirmed
experimentally in beautiful studies with transcripts of synthetic DNAs
of defined sequences by John Carbon and Paul Berg (31) and N. Gupta and
Gobind Khorana (32). Paul has described our personal interactions that
led to these successful in vitro studies (33).
| |
Opening Pandora's Box |
Following completion of our colinearity studies and our foray
into deducing the genetic code, there were many unsolved biological problems begging for our attention. The course I followed was conservative; I decided to exploit the knowledge we had recently gathered and attempt to deducing answer what I considered the next set
of important questions including the following. How does each trp
enzyme catalyze its respective reaction? What are the three-dimensional
structures of the trp enzymes, and how are they related?
What are the advantages of forming two enzyme complexes, each
containing two different trp polypeptides? What is the purpose of
producing two bifunctional trp polypeptides? What is the significance, if any, of the order and organization of the trp genes in the trp
operon? What is the explanation for the polar effect of nonsense mutations on downstream gene expression, and what is its significance? What are the important features of transcription, translation, and
mRNA degradation for the trp operon of E. coli? What
were the ancestral sources of the genes specifying the trp biosynthetic enzymes? Addressing one of these biochemical questions, graduate student Tom Creighton analyzed the subunit structure of the tryptophan synthetase enzyme complex in the mid-1960s. In a collaborative study
with Michel Goldberg and Robert Baldwin of the Biochemistry Department
at Stanford, they concluded that this enzyme complex has an
structure (where isTrpA and is TrpB) with alone existing as a monomer and alone as a 2 dimer (34).
Convinced that structural information was essential if we were to
provide a thorough understanding of this enzyme's action, Ulf Henning grew beautiful crystals of the E. coli chain hoping they
would be suitable for crystallographic analysis. In addition, I spent a
summer at the University of California in San Diego exploring with
members of Joe Kraut's group the possibility of growing chain
crystals satisfactory for structure determination. This approach was
pursued by Tom Creighton when he moved to Yale. He had some success,
but unfortunately satisfactory crystals of the tryptophan synthase subunit of E. coli could not be grown reproducibly. On a
related project, John Hardman of my group initiated studies on the
three cysteine residues in the TrpA polypeptide that we thought were
essential. His findings on substrate protection of these three
cysteines were provocative, but it was evident that without the
three-dimensional structure of the protein for reference, these active
site studies would be inconclusive. I therefore discontinued work on
this project. As I mentioned, the structure of the tryptophan synthase enzyme complex from Salmonella was
eventually solved by Craig Hyde, Edith Miles, David Davies, and their
co-workers (15). The structural information they provided served as an invaluable resource for many years, allowing crucial questions to be
answered, such as how do the two active sites in the enzyme complex
catalyze their respective reactions and how are these sites
cross-activated by substrate binding (16, 35).
Ted Cox took a broader view of the consequences of mutations and
questioned their impact on organism well being and survival. While with
me he began his studies with the mutT mutator gene of
E. coli. In 1967 we showed that mutT causes AT to
CG mutations preferentially and that continued cultivation of strains
with mutT led to a uniform shift in the base composition of
their total DNA (36). The changes he detected represented about a
0.2-0.5% increase in GC composition. This observation raised
additional questions. What fraction of the residues in each
protein is essential? What fraction of the base pairs in the genome of
E. coli can be changed without having serious consequences? I
decided not to address these questions at this time.
| |
Turning Our Attention to Organization and Expression of the
trp Operon |
In the mid 1960s the features of the trp operon of
E. coli that contributed to its expression were poorly
understood. The order of the five genes in the operon had been
established, but very little was known about operon transcription or
how trp mRNA translation and degradation proceeded or
how these processes were regulated. These basic questions were exciting
to young molecular microbiologists, and new members of my group were
eager to address one or more of these problems. My co-workers on these
subjects from the mid-1960s to the early 1970s were Ron Somerville, Dan Morse, Ray Mosteller, Ron Baker, Robert Baker, Jack Rose, Jun Ito,
Fumio Imamoto, Ethel Jackson, Jes Forchhammer, and Sota Hiraga. Of
significant aid in our mRNA studies was the use of a temperate bacteriophage, 
80, characterized by A. Matsushiro. This phage genome
integrates adjacent to the trp operon, allowing one to obtain improperly excised transducing phage that carry different segments of the trp operon. The DNAs of these trp
transducing phage could then be used to detect and measure the relative
amounts of labeled mRNA derived from any segment of the operon. A
very important, but unrelated project, was carried out with this phage by Naomi Franklin, who was then in my laboratory, with Bill Dove at our
Medical School. They provided genetic evidence indicating that during
lysogenization the 80 genome is inserted into the bacterial
chromosome. I believe this was the first experimental evidence
supporting the Campbell integration model of lysogenization (37).
Using the isolated DNA of trp transducing phage bearing
different segments of the trp operon, RNA hybridization data
were gathered for different genes of the operon. It was shown that the
operon specifies a single polycistronic trp mRNA
encoding all five of the trp polypeptides and that the
transcript was translated as it was being synthesized. It was also
observed that nascent trp mRNA was generally attacked
before its synthesis was completed. Thus most trp
transcripts isolated from growing cultures were less than full length
(38). The last coding region of the trp operon transcript,
trpA mRNA, was found to be degraded in the 3' to 5'
direction (39). Most nonsense mutations in the first four genes of the
operon had a negative, polar effect on downstream gene expression,
reducing both trp mRNA and protein levels for the
downstream genes (40). This "polarity" was a common observation with many bacterial systems. We also found that the untranslated mRNA segment immediately downstream of each introduced nonsense codon was particularly labile (41), consistent with Rho-mediated transcription termination in the untranslated region of the messenger and 3' to 5' degradation of each untranslated mRNA segment. Ron Somerville observed continued synthesis of the TrpA polypeptide, but
not the TrpB polypeptide, upon prolonged tryptophan starvation (42).
His findings were consistent with the presence of a single Trp residue
in TrpB but none in TrpA (43). The location of the internal promoter
within the trp operon, previously identified by Bauerle and
Margolin in the Salmonella trp operon (44), was determined for E. coli by Ethel Jackson of my group by
preparing and examining internal deletions in the operon (45).
Ultimately its nucleotide sequence (for E. coli) was
established by Terry Platt's group when Terry had his own laboratory
(46). In other studies with the TrpA protein, Dave Jackson observed
that he could complement (restore activity to) a mutant TrpA
polypeptide in vitro by unfolding and refolding the
polypeptide in the presence of a second mutant TrpA polypeptide that
had an amino acid change elsewhere in the protein (47). Refolding of a
mixture of mutant polypeptides allowed this normally monomeric protein
to occasionally form an active dimeric species. Restoration of enzyme
activity also was observed upon refolding a mutant polypeptide in the
presence of a short fragment of wild type polypeptide that corresponds to the mutated segment (47). A model has been proposed explaining these
examples of in vitro complementation (16). These studies suggested interesting approaches that could be used in studying the
mechanism of protein folding.
| |
On to Operon Regulation |
Despite these advances, we had not yet begun to address what
was becoming the most challenging question for most bacterial physiologists. How is transcription of your operon
regulated? In early regulatory studies with the trp
operon of E. coli, Georges Cohen and Francois Jacob
identified a presumed repressor locus, trpR, that appeared
to negatively regulate expression of the trp operon. In the
early 1960s the only additional regulatory observation that concerned
tryptophan biosynthesis was the finding that the enzyme catalyzing the
initial reaction in the pathway, anthranilate synthase, was
feedback-inhibited by tryptophan. Feedback inhibition of the enzyme
performing the first reaction in a pathway is common to most
biosynthetic pathways. At this time we were reasonably comfortable with
the belief that repression plus feedback inhibition for the
trp operon could deal with all the regulatory needs of the
bacterium. To analyze repression more thoroughly, Cathy Squires and
Jack Rose of my group partially purified the trp repressor and (with the aid of Goeffrey Zubay and H. L. Yang) performed in
vitro analyses showing that the trp repressor is
tryptophan-activated and that the repressor does inhibit transcription
initiation at the trp operon promoter. Follow-up studies by
Jack Rose, Cathy Squires, Frank Lee, Rick Kelley, George Bennett, and
Rob Gunsalus developed the trp repressor-trp
operator into an excellent experimental system. They showed that the
repressor is a dimer, that it has two helix-turn-helix DNA binding
domains, and that crucial base pairs in the palindromic trp
operator are required for repressor binding (48-52). With the aid of
Andrzej Joachimiak from Paul Sigler's group, the trp
repressor was purified and initially characterized. Sigler's group
then initiated their elegant studies culminating in determination of
the three-dimensional structures of the trp aporepressor,
the tryptophan-activated trp repressor, and the trp repressor-trp operator complex (53). Their
studies represent one of the most thorough analyses of repressor
action. Oleg Jardetzky's group at Stanford, using NMR
technology, also established the structures of the aporepressor,
repressor, and repressor-operator complex (54). Our parallel in
vivo studies revealed that the activated trp repressor
reduces transcription initiation at the trp operon
promoter/operator region about 80-fold (55). The trp
repressor was also shown to regulate transcription initiation at the
promoter/operators of several other operons concerned with tryptophan
metabolism, in addition to being autoregulatory. Several of these
operator regions have multiple repressor binding sites; for example,
the trp operon operator region has three (56, 57). Excellent
studies on these and other features of trp repressor action
have been performed by scientists at other institutions: Janette Carey,
C. Robert Matthews, C. L. Lawson, K. S. Matthews, C. A. Royer,
C. H. Arrowsmith, and others.
| |
A Surprise: the trp Operon Is Also Regulated by Transcription
Attenuation! |
In the early 1970s we were well aware of the findings by other
groups who were conducting regulatory studies with amino acid biosynthetic operons of bacteria. The experimental results of Bruce
Ames and his co-workers at the University of California, Berkeley, were
of particular interest to us because the his operon of
Salmonella they were studying and our trp operon
had many similarities. Ames showed that transcription of the
his operon was not regulated by a histidine-responsive
his repressor; rather, histidinyl-tRNA was implicated as the
molecule that was sensed in the regulatory decision (58). Furthermore,
the leader region of the his operon, not its promoter,
appeared to be the site of regulation. Graduate student Ford Doolittle
of my group was persuaded to consider these findings seriously, and he
performed a series of regulatory studies with slightly defective
E. coli tryptophanyl-tRNA synthetase mutants. His results
demonstrated that tryptophanyl-tRNA is not involved in trp
repressor action; thus his findings put our concerns to rest, at least
for the moment (59). However, measurements of trp mRNA
levels carried out during this period by Ron Baker of my group
suggested that there may be a second regulatory mechanism, distinct
from repression, that regulates transcription of the trp
operon of E. coli. Baker observed that mutants lacking a
functional trp repressor still responded to tryptophan
starvation by increasing their rate of synthesis of trp
mRNA. Consistent with this observation was the finding by Fumio
Imamoto, then back in his own laboratory in Japan, that transcription
in progress in the initial segment of the trp operon was
stopped prematurely upon addition of tryptophan to a tryptophan-starved
culture. We wondered: what is the significance of these
regulatory findings?
| |
Explaining Transcription Attenuation |
In the early 1970s Ethel Jackson made the key observation that
convinced me to search for a regulatory mechanism distinct from
repression that regulates transcription of the trp operon (60). As mentioned, Ethel developed a procedure that allowed her to
isolate deletions with both end points within the trp
operon. Her initial objective was locating the internal promoter
precisely. During these studies she made the unexpected observation
that a class of internal deletions with one end point in the leader region of the operon, the region just following the promoter and before
the first structural gene, trpE, increased operon expression 6-fold. This increase also was observed in a repressor minus strain! This suggested that there may be a second regulatory site, possibly a
site of regulated transcription termination, that can influence trp operon expression
TOP
INTRODUCTION
Reflections: Questions,...