From the Department of Biological Sciences, Stanford University, Stanford, California 94305
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.
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.
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, 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 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.
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 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!
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 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.
INTRODUCTION
Reflections: Questions, Answers, and More Questions
The One Gene-One Enzyme Relationship
-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.
. 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.)
View larger version (16K):
[in a new window]
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).
Changing my Experimental Organism
2
2 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.
Proving or Disproving Gene-Enzyme Colinearity
), 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.
View larger version (12K):
[in a new window]
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).
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.
![]() |
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).
![]() |
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 (60). At about the same time, A. Kasai, at Johns Hopkins University, was performing similar analyses of the effects of deletions that ended in the leader region of the his operon of Salmonella. Kasai also concluded
that the his operon leader region may contain a regulated
site of transcription termination (61). He introduced the term
"transcription attenuation" to describe the mechanism of
transcription regulation that presumably occurs at this site. I adopted
this term in our studies with the trp operon because I felt
it was entirely appropriate.
![]() |
Attenuation Proves to Be a Complex, Multistep Process |
---|
In the early and mid-1970s most members of my group were studying features of the trp operon and tryptophan metabolism that we believed contributed to operon expression or regulation. We did not appreciate that each was analyzing an event that was crucial to transcription attenuation. The trp operon leader region (the genetic segment responsible for transcription attenuation) was isolated and characterized. It is ~160 bp in length and is located between the promoter and the first major structural gene of the operon. This "leader region" was sequenced, first as RNA, and then, when DNA sequencing technology became available, as DNA. This sequence raised several new questions requiring our immediate attention. What features of the leader region are responsible for transcription attenuation? Is tryptophan or tRNATrp the signal that is recognized during attenuation in the trp operon? Does the leader region sequence provide any clues that would help us to explain how one of these molecules could act as a regulatory signal? A potential transcription termination site was located in the leader region just before trpE. It had all the features now ascribed to intrinsic transcription termination sites. The members of my group who performed these initial studies were: Kevin Bertrand, Craig Squires, Cathy Squires, Frank Lee, Morley Bronson, Terry Platt, Laurence Korn, George Bennett, Iwona Stroynowski, and Giuseppe Miozzari. Terry Platt had been attempting to identify all the ribosome binding sites in trp operon mRNA. He detected one ribosome binding site that was unanticipated; it was located in the leader segment of the transcript. This was an exciting discovery because this site was associated with a 14-residue coding region with two adjacent tryptophan codons. This coding region was located just prior to the terminator sequence. To explore the function of this leader peptide coding region we added the following questions to our "list" of those we felt must be addressed. What is the role of the transcript's leader peptide coding region, is it regulatory, and does it allow regulation of transcription termination? If it is regulatory, do the Trp codons in the leader peptide coding participate in the regulatory decision? Is the regulatory signal that is sensed uncharged tRNATrp? It was evident we were dealing with unfamiliar events in a complex process. Fortunately for us, previous findings with the his operon addressed several of these questions; therefore they were extremely helpful. In retrospect, every member of my group contributed to answers to one or more of these questions. We were aided in these studies by Larry Soll, a former student of Paul Berg, who was then at the University of Colorado; Larry performed some of the crucial early experiments implicating tRNATrp as the regulatory signal (62). Dan Morse, after leaving my laboratory, independently contributed findings establishing the role of uncharged tRNATrp in trp operon attenuation (63). It was becoming clear that transcription attenuation in the trp operon involved several sequential events, each dependent upon specific sequences in the transcript of the leader region.
Of particular significance during this period was the finding by graduate student Frank Lee that the trp leader transcript could fold to form alternative hairpin structures, each of which plays an essential role in determining whether transcription termination will occur (64). One RNA hairpin serves as a transcription terminator; it directs RNA polymerase to terminate transcription. The second, alternative RNA hairpin functions as an antiterminator. Inspection of the sequence revealed that prior formation of the antiterminator would prevent formation of the terminator. Which of the alternative hairpin structures would form would depend on the cell's ability to translate the two Trp codons in the 14-residue leader peptide coding region. When these two tryptophan codons are translated, the antiterminator would not form; this would allow the terminator to form and terminate transcription (65, 66). When cells are deficient in charged tRNATrp the translating ribosome would stall at one of the Trp codons. This stalling would promote antiterminator formation, which would then prevent formation of the terminator (65, 66).
Studies over the past 30 years have shown that transcription
attenuation is a common regulatory process; variations are used by many
bacterial species and their viruses (67). Several transcription attenuation mechanisms are described in a recent review by Henkin and
myself (68). In the earliest studied example of regulation by
transcription termination/antitermination, the N protein of bacteriophage was shown to prevent Rho-dependent
transcription termination during transcription of a region of the phage
genome. Attenuation in the his operon of
Salmonella, as mentioned, also was an early studied example;
most of its features closely resemble those of attenuation in the
E. coli trp operon. More recently it has been
learned that in addition to ribosome and protein-mediated transcription
attenuation decisions, uncharged tRNA (69) and various metabolites can
interact directly with leader RNA and regulate transcription
termination (70). For example, many of the genes encoding
aminoacyl-tRNA synthetases in B. subtilis and other Gram-positive bacteria have been shown by Frank Grundy and Tina
Henkin and their co-workers to be regulated by direct tRNA-mediated transcription attenuation (71). There are related translational examples where translation of an upstream mRNA coding region
influences translation initiation at the adjacent downstream coding
region (72). Often translation initiation at the downstream coding region is blocked by an appropriate RNA secondary structure; an event
occurring during translation of the upstream coding region, such as
chloramphenicol binding to the translating ribosome, then exposes the
downstream translation initiation region to ribosome loading and
translation initiation (72).
Following this initial period of our investigations on transcription
attenuation, it was obvious that we would have to establish the role of
each segment of the trp leader transcript and explain the
many events participating in this process. Gerard Zurawski, George
Stauffer, and Dirk Elseviers, and sabbatical visitors Keith Brown and
Dale Oxender performed important studies that provided a thorough
understanding of many of the features of transcription attenuation in
the trp operon of E. coli (65, 66). One obvious, crucial concern that we had not yet addressed was as follows. How
are transcription and translation of the trp leader region coordinated
and coupled, as they must be if translation of the leader peptide
coding region is to serve as the decision-making event regulating
transcription termination? Postdoctoral fellows Malcolm Winkler,
Bob Fisher, Bob Landick, and Jannette Carey answered this question.
They established the role of a third hairpin structure that can form in
the trp leader transcript, a structure that precedes and is
an alternative to the antiterminator. This structure, which also serves
as an anti-antiterminator, causes the transcribing RNA polymerase to
pause during transcription of the leader region (73-76). This pause
allows sufficient time for a ribosome to bind to and initiate
translation of the leader peptide coding region. Landick and Carey in
fact showed that it is this translating ribosome that releases the
paused RNA polymerase, allowing transcription and translation to
proceed simultaneously (77). This coupling is essential to allow
charged or uncharged tRNATrp to be recognized by the
translating ribosome and serve as the regulatory signal. Progress has
been made in explaining polymerase-transcript interactions that are
responsible for transcription pausing, thanks to thorough studies on
this subject by Bob Landick and his group (78) and detailed structural
analyses on RNA polymerases and their action provided by Roger
Kornberg, Seth Darst, and their co-workers. A simplified view of most
of the stages in regulation by transcription attenuation in the
trp operon of E. coli is presented in Fig.
3.
|
In vivo analyses were also performed to assess the relative contributions of repression and transcription termination in regulating trp operon expression (55). We concluded that repression regulates transcription initiation in the trp operon about 80-fold, with repression at a minimum during growth with little or no tryptophan. In contrast, transcription attenuation in the trp operon of E. coli allows only 6-fold regulation, with termination being relieved only when cells are virtually depleted of charged tRNATrp. The relative insensitivity of attenuation regulation of the trp operon to the accumulation of uncharged tRNATrp reflects the presence of only two Trp codons in the trp leader peptide coding region. In the his operon of Salmonella, for example, where attenuation is the major transcription regulatory mechanism, there are seven contiguous His codons in the leader peptide coding region. This organization makes the his operon particularly sensitive to a deficiency of charged tRNAHis.
DNA microarray analyses have been performed with wild type E. coli and several regulatory mutants under a variety of growth conditions that influence tryptophan metabolism (79). In general the
changes in mRNA abundance observed are consistent both
qualitatively and quantitatively with expectations based on years of
studies of tryptophan metabolism. As expected, many indirect effects
were also observed.
![]() |
Miscellaneous Important Developments |
---|
A major advance in conventional cloning was the development of
plasmid ColE1 for this purpose by Don Helinski, Herb Boyer, and their
co-workers in the early 1970s. We provided the trp operon for these studies and analyzed its expression in their classic plasmid
cloning/overexpression paper (80). When DNA cloning and sequencing
procedures became available, my group collaborated with several of my
former students in determining the complete 7000-base pair sequence of
the trp operon of E. coli; this sequence was
published in 1981 (43). Inspection of the nucleotide sequence of the
operon revealed many unsuspected features. Among these was the presence
of overlapping stop and start codons, UGAUG, joining trpE
and trpD, and trpB and trpA (43). This
punctuation arrangement was intriguing because we already knew that
both the TrpE and TrpD polypeptides, and the TrpB and TrpA
polypeptides, associate to form enzyme complexes. We wondered,
what is the significance of these stop/start overlaps?
Do they allow some form of translational coupling that ensures that the
cell synthesizes equal numbers of polypeptides that will form an enzyme
complex? Dan Oppenheim and Anath Das addressed these questions and
concluded that translation of these adjacent coding regions is coupled,
i.e. equal numbers of the two polypeptides encoded by
adjacent regions are produced only when the upstream coding region is
translated to completion at its overlapping stop codon (81, 82).
Translation initiation and termination obviously are very complex
processes; transcript sequences can have profound effects on these events.
![]() |
Studies on Regulation of Tryptophan Degradation |
---|
E. coli and many other bacteria have the ability to degrade tryptophan. They produce the enzyme tryptophanase, which degrades tryptophan to indole, pyruvate, and ammonia. Pyruvate and ammonia can be used as carbon and nitrogen sources. Indole's role, other than serving as a tryptophan precursor, is not clear, although recent evidence suggests that it may act as a volatile signal molecule during biofilm formation and quorum sensing (83, 84). The latter observations raise an additional unexpected question. Is the purpose of the tunnel connecting the two active sites of tryptophan synthase to prevent biosynthetic indole from escaping into the environment?
Because degradation of tryptophan would be expected to influence
regulation of its synthesis, I decided in the early 1980s that it was
essential that we thoroughly investigate the tryptophanase (tna) operon and how it is regulated. Our initial questions
were as follows. How is the tna operon organized? How is this
operon regulated? What are the effects of tna operon expression on trp operon expression and regulation? Studies on these questions were initiated by Mike Deeley; he was followed on this project by Valley Stewart, Paul Gollnick, Kurt Gish, Ajith Kamath, Vincent Konan, and
most recently, Feng Gong. The tna operon of E. coli has two structural genes, one encoding tryptophanase and the
second specifying a tryptophan permease. Transcription initiation in
this operon had been shown by others to be regulated by catabolite
repression. In our investigations we discovered that transcription of
the structural genes of this operon is regulated by a novel mechanism of transcription attenuation. This mechanism is based on features of
the nucleotide sequence of the operon's ~300-bp leader region. Tryptophan is the signal molecule that leads to relief from
transcription termination. When cultures are growing without excess
tryptophan, Rho factor binds to the nascent tna operon
leader transcript. Bound Rho then contacts the transcribing polymerase
that is paused at one of several pause sites in the leader region, and
it instructs it to terminate transcription (85, 86). If cultures are
growing with high levels of tryptophan, Rho factor's ability to bind
to the leader RNA is prevented. Therefore the paused polymerase resumes transcription into the structural genes of the operon (85, 86). Synthesis of a 24-residue tryptophan-containing leader peptide, TnaC,
as well as high levels of free tryptophan are required for induction
(86). It is thought that the combined action of the nascent uncleaved
TnaC-peptidyl-tRNA and bound tryptophan inhibits peptidyl transferase
cleavage of the TnaC-peptidyl-tRNA (87, 88). The uncleaved
TnaC-peptidyl-tRNA therefore remains associated with the translating
ribosome, preventing it from dissociating from the transcript. The
stalled ribosome then blocks Rho factor's access to the transcript,
thereby allowing the paused polymerase to resume transcription (87).
Recent studies suggest that the tryptophan binding/induction site may
be the site normally occupied by the aminoacyl moiety of a charged tRNA
during translation (88). These findings raise challenging questions
about the functional flexibility of the ribosome, questions that become
more interesting when they are related to recent exciting structural
studies with the ribosome. What are the structural features of
the tryptophan binding site created in the ribosome? How does the
tryptophan residue at position 12 of the leader peptide create or
modify this binding site? How does bound tryptophan inhibit
peptidyl transferase? The presence of active tryptophanase in a
growing culture reduces the tryptophan concentration, which increases trp operon expression (89).
![]() |
Evolutionary Issues |
---|
As our gene structure and function studies progressed, many evolutionary questions arose. Do other organisms use the same genes, proteins, operon organization, and regulatory processes as E. coli in performing tryptophan biosynthesis and its regulation? Can homologous segments of a trp polypeptide from two species be exchanged without loss of enzyme activity? What were the ancestors of the present day genes and proteins of tryptophan biosynthesis? Members of my group who addressed these questions were Steven Li, Iwona Stroynowski, Bill Schneider, Brian Nichols, Richard Denney, Mike Manson, Joan Hanlon, Eric Selker, and Giuseppe Miozarri. To begin with, the trp genes of many organisms were cloned and partially or completely sequenced; the sequences and genetic locations then were compared. Many other laboratories provided comparable information for their favorite genes and operons. Comparative studies with the trp genes revealed that all organisms that synthesize tryptophan use the same seven catalytic enzyme domains. The enzymes of the tryptophan biosynthetic pathway therefore probably evolved just once. However, within each polypeptide, when one compares different species, there is appreciable sequence variation. This is typical for most protein evolutionary comparisons. In one beautiful study performed in my laboratory by Bill Schneider and Brian Nichols, segments of the TrpAs of E. coli and Salmonella were exchanged, generating recombinant TrpA polypeptides. All the recombinant TrpA proteins produced were fully functional despite the fact that 40 of the 268 amino acid residues in the parental homologous TrpA proteins differ (90). This result implies that most of these amino acid differences are tolerable when inserted individually or in clusters.
Evolutionary comparisons of the organization of the seven trp genes in different species revealed appreciable variation. In some species the trp genes are split in several operons, and often two or more trp genes are fused to form multifunctional enzymes. Regulatory mechanisms also vary considerably, possibly reflecting differences in operon organization and participation of one or more pathway intermediates in a second pathway. Nucleotide sequence divergence for the trp genes correlated well with predictions of species relatedness based on analyses of ribosomal RNA sequences although there are some hints of horizontal transfer.
Understandably, I was particularly interested in knowing whether the features of repression and transcription attenuation observed with the trp operon of E. coli are conserved in unrelated species. Among the enteric bacteria we examined the major features were retained, although there was considerable leader sequence variation, presumably reflecting slightly different species-specific objectives (91). Mitzi Kuroda of my group initiated comparative regulatory studies with the trp operon of a second well studied prokaryote, B. subtilis. We were joined in this effort by Dennis Henner and his group at Genentech when we learned that he too was concerned with this problem. We already knew that there were significant differences in trp gene and operon organization in B. subtilis versus E. coli. B. subtilis has seven distinct trp genes, only six of which are clustered as a trp operon. Furthermore the six-gene trp operon is located within an aromatic supraoperon, which has three additional upstream genes and three additional downstream genes, each concerned with some aspect of aromatic amino acid metabolism (92). The seventh trp gene, trpG, is located in the folate biosynthetic operon (92). Its location is logical because its polypeptide product, TrpG, is a glutamine amidotransferase that is a component of two similar enzyme complexes, one catalyzing para-aminobenzoate synthesis in the folate pathway and the second catalyzing ortho-aminobenzoate (anthranilate) formation in the tryptophan pathway. The studies by Mitzi Kuroda and the Henner group were followed by investigations in my laboratory by Paul Gollnick, Paul Babitzke, Joe Sarsero, Enrique Merino, and most recently, by Angela Valbuzzi and Guang-nan Chen. The basic features of attenuation regulation of trp operon expression in B. subtilis were established; they were quite different from those used for the trp operon of E. coli. Although alternative antiterminator and terminator hairpin structures also participate in the transcription termination decision, an 11-subunit tryptophan-activated RNA-binding protein, named TRAP, is used by B. subtilis to disrupt the antiterminator and promote terminator formation (93-95). We selected TRAP as the name for this protein because it is a trp RNA-binding attenuation protein (93). Tryptophan-activated TRAP also binds trpG mRNA and several other RNAs concerned with tryptophan metabolism. The TRAP binding sites in these transcripts all overlap translation start sites; thus TRAP binding also regulates translation initiation (93). Furthermore, TRAP binding to trp operon leader RNA indirectly regulates translation initiation at the trpE start site (96). A most interesting recent discovery in my laboratory is that B. subtilis contains a previously unidentified regulatory operon, rtpA-ycbK, that is designed to sense (and respond to) uncharged tRNATrp (97). We named the rtpA protein AT (Anti-TRAP) because it is designed to bind to and inactivate tryptophan-activated TRAP (98, 99). Transcription of the rtpA-ycbK operon is regulated by tRNATrp-mediated transcription attenuation by the T box antitermination mechanism discovered by Grundy and Henkin (69). We recently observed that uncharged tRNATrp accumulation has a second regulatory effect on the rtpA-ycbK operon; it increases translation of rtpA, thereby providing higher levels of AT protein (G. Chen and C. Yanofsky, manuscript in preparation). Thus B. subtilis employs two independent mechanisms of sensing uncharged tRNATrp in this operon; one is transcriptional and the second translational; both regulate AT synthesis (G. Chen and C. Yanofsky, manuscript in preparation). DNA microarray analyses have also been performed with wild type and regulatory mutants of B. subtilis to analyze the total genome's transcriptional response during growth under nutritional conditions that affect tryptophan metabolism (R. M. Berka, X. Cui, and C. Yanofsky, manuscript in preparation). The genes we expected to respond did; however, many additional genes responded comparably, suggesting that their expression is closely tied to the genes involved in tryptophan synthesis.
The knowledge we have gathered in our studies on tryptophan metabolism
in E. coli and B. subtilis raise the very
tough "why" question. Why do E. coli and B. subtilis use such
dissimilar mechanisms to sense tryptophan and tryptophan tRNA as
regulatory signals? I would love to know the answer!
![]() |
Enzyme Structural Questions |
---|
One particular set of challenging questions was always on my
wish list, but I left these questions for other scientists to answer.
What are the three-dimensional structures of the seven protein
domains required for tryptophan synthesis? Are any of these
domains evolutionarily related? What are the
likely ancestral sources of the seven catalytic domains? I am
delighted to report that the three-dimensional structures for all seven
protein domains required for tryptophan synthesis have been determined.
This knowledge should permit investigators to consider structural as
well as catalytic issues when attempting to deduce possible
evolutionary origins for these domains. Interestingly, the structures
of three of the tryptophan pathway enzymes are 8-fold barrels.
Particularly exciting in this regard is the recent demonstration that
an enzyme catalyzing a reaction in histidine biosynthesis (a reaction
similar to one catalyzed by an enzyme of the tryptophan pathway) was
converted into an active trp enzyme by introducing a single
amino acid change (100). Given the extraordinary wealth of information
being provided by sequence analyses, evolutionary exploration of enzyme
origins should be an interesting subject for future investigations.
![]() |
Returning to Neurospora |
---|
Whenever I selected a research project for one of my graduate
students I was well aware that this individual might prefer to work on
some other problem as a member of my group. Eric Selker, an exceptional
graduate student who joined me in the late 1970s, decided that the
project I had assigned him, characterizing the genes of the
trp operon of Salmonella typhimurium, would not
break new ground, and therefore he preferred to work on a project that was more challenging. He proposed reintroducing N. crassa
into my laboratory as an experimental organism. As I recall, I resisted Eric's proposal to switch his project only modestly. I knew this organism well, and most importantly, one of my closest colleagues, David Perkins, whose laboratory is just down the hall, was a major contributor to Neurospora research. Eric convinced me that
the time had come to apply the procedures, technology, and concepts developed in studies with bacteria and yeast to the superb experimental eukaryote selected by Beadle and Tatum as their experimental organism. At a minimum, I thought, we should be able to compare the genes and
proteins of tryptophan metabolism and their regulation in N. crassa with those in E. coli. A few years later a
second bright graduate student, Vivian Berlin, also with my approval,
switched from her initial bacterial studies to apply modern molecular
approaches in analyzing an excellent model developmental process in
Neurospora, asexual spore formation. Many talented graduate
students and postdoctoral fellows subsequently joined my group to
perform fungal studies. Their work greatly improved the technology that
could be applied in investigations with Neurospora. Most of
these individuals, after leaving my group, continued to make
significant scientific contributions in fungal biochemistry and genetics.
![]() |
My Treasures |
---|
Reflecting on what I would consider our two most impressive contributions, I would select proving gene-protein colinearity and determining the stages and features of regulation by transcription attenuation. The first required our identifying the amino acid changes in a set of TrpA mutant proteins and comparing the relative locations of these amino acid changes with the order of the corresponding mutational changes on the genetic map of the trpA gene. Essentially, we verified a relationship, which, at the time, we believed existed. Transcription attenuation, by contrast, was a poorly understood process initially thought to be used only rarely. We were required to break new ground and perform step by step analyses of the roles played by tandem overlapping segments of a transcript, as well as explaining how ribosome stalling at either of two Trp codons selects between alternative RNA structures. I had no reason to suspect that transcription attenuation was such a common regulatory strategy or that so many different mechanisms of attenuation existed. I have illustrated in this article how the answers we obtained while focusing on some specific questions invariably raised new unanswered questions. More often than not, these questions were so challenging they could not be ignored.
Despite the enormous satisfaction I feel personally from what we have
accomplished scientifically, I believe my greatest pleasure in
practicing science has come from the give and take of daily interactions with members of my group and from thoughtful and stimulating discussions with fellow scientists. My journey in science
has been great fun! I was very fortunate to have had Dave Bonner as my
mentor and lucky that I "grew up" with a wonderful group of smart
graduate students. Learning biochemistry from Joseph Fruton was an
extraordinary experience. At Western Reserve, Howard Gest, Bob
Greenberg, Abe Stavitsky, and John Spizizen were all special friends
who contributed to my development. At Stanford, because of our personal
friendship and frequent discussions, Paul Berg put his stamp of
approval on virtually everything I have done. I have had many other
close friends and colleagues at Stanford, including Dave Perkins, Don
Kennedy, Norm Wessells, Paul Ehrlich, Bob Schimke, Phil Hanawalt, Bob
Simoni, Dale Kaiser, Dave Hogness, Lucy Shapiro, and Bob Lehman.
Scientists at other institutions, some of whom spent a sabbatical in my
laboratory, also were great friends, including Howard Zalkin, Frank
Gibson, Dale Oxender, Ron Bauerle, Kasper Kirschner, Edith Miles, Stan
Mills, Michael Chamberlin, and Paul Sigler. Stan Prusiner, an
outstanding scientist with completely different interests, became a
very close friend. Finally, throughout the past 40+ years of my career
Arthur Kornberg's wisdom and commitment have served as models guiding
my behavior. There are many other "treasured" individuals who I did
not get to mention in this article; I thank you all.
![]() |
ACKNOWLEDGEMENTS |
---|
I am extremely grateful to Howard Gest, Cathy Squires, Edith Miles, and Robert Simoni for reading a draft of this manuscript and providing many helpful comments. I would also like to acknowledge the support provided for my research activities by the National Institutes of Health, National Science Foundation, American Heart Association, and American Cancer Society. I have previously written a biographical article describing many of our accomplishments (101).
![]() |
FOOTNOTES |
---|
Published, JBC Papers in Press, January 29, 2003, DOI 10.1074/jbc.X200012200
Address correspondence to: yanofsky{at}cmgm.stanford.edu.
![]() |
REFERENCES |
---|
1. | Beadle, G. W., and Tatum, E. L. (1941) Genetic control of biochemical reactions in Neurospora. Proc. Natl. Acad. Sci. U. S. A. 27, 499-506 |
2. |
Hershey, A. D.,
and Chase, M.
(1952)
Independent functions of viral protein and nucleic acids in growth of bacteriophage.
J. Gen. Physiol.
36,
39-56 |
3. | Avery, O. T., MacLeod, C. M., and McCarty, M. (1944) Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. Exp. Med. 79, 137-157 |
4. | Watson, J. D., and Crick, F. H. C. (1953) Molecular structure of nucleic acids. Nature 171, 737-738 |
5. | Benzer, S. (1957) The elementary units of heredity. In The Chemical Basis of Heredity , pp. 70-93, Johns Hopkins University Press, Baltimore, MD |
6. | Sanger, F. (1952) The arrangement of amino acids in proteins. Adv. Protein Chem. 7, 1-28 |
7. |
Umbreit, W. W.,
Wood, W. A.,
and Gunsalus, I. C.
(1946)
The activity of pyridoxal phosphate in tryptophan formation by cell-free enzyme preparations.
J. Biol. Chem.
165,
731-732 |
8. |
Mitchell, H. K.,
and Lein, J.
(1948)
A Neurospora mutant deficient in the enzymatic synthesis of tryptophan.
J. Biol. Chem.
175,
481-482 |
9. | Hogness, D. S., and Mitchell, H. K. (1954) Genetic factors influencing the activity of tryptophan desmolase in Neurospora crassa. J. Gen. Microbiol. 11, 401-411[Medline] [Order article via Infotrieve] |
10. | Yanofsky, C. (1952) The effects of gene change on tryptophan desmolase formation. Proc. Natl. Acad. Sci. U. S. A. 38, 215-226 |
11. | Suskind, S. R., Yanofsky, C., and Bonner, D. M. (1955) Allelic strains of Neurospora lacking tryptophan synthetase: a preliminary immunochemical characterization. Proc. Natl. Acad. Sci. U. S. A. 41, 577-582 |
12. | Yanofsky, C. (1956) Gene interactions in enzyme synthesis. Henry Ford Hospital International Symposium: Enzymes, Units of Biological Structure and Function , pp. 147-160, Academic Press Inc., New York |
13. | Yanofsky, C. (1954) The absence of a tryptophan-niacin relationship in Escherichia coli and Bacillus subtilis. J. Bacteriol. 68, 577-584[Medline] [Order article via Infotrieve] |
14. | Yanofsky, C., and Rachmeler, M. (1958) The exclusion of free indole as an intermediate in the biosynthesis of tryptophan in Neurospora crassa. Biochim. Biophys. Acta 28, 641-642 |
15. |
Hyde, C. C.,
Ahmed, S. A.,
Padlan, E. A.,
Miles, E. W.,
and Davies, D. R.
(1988)
Three-dimensional structure of the tryptophan synthase ![]() ![]() |
16. | Miles, E. W. (1995) in Tryptophan synthase: structure, function, and protein engineering. In Subcellular Biochemistry, Proteins: Structure, Function, and Protein Engineering (Biswas, B. B. , and Roy, S., eds), Vol. 24 , pp. 207-254, Plenum Press, New York |
17. | Crawford, I. P., and Yanofsky, C. (1958) On the separation of the tryptophan synthetase of Escherichia coli into two protein components. Proc. Natl. Acad. Sci. U. S. A. 44, 1161-1170 |
18. | Ingram, V. M. (1958) Abnormal human hemoglobins. 1. The comparison of normal human and sickle-cell hemoglobins by fingerprinting. Biochim. Biophys. Acta 28, 539-545 |
19. | Yanofsky, C., and Lennox, E. S. (1958) Transduction and recombination study of linkage relationships among the genes controlling tryptophan synthesis in Escherichia coli. Virology 8, 425-447 |
20. | Yanofsky, C. (1963) Discussion following article by W. Gilbert: Protein Synthesis in Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 28, 296-297 |
21. | Yanofsky, C., Carlton, B. C., Guest, J. R., Helinski, D. R., and Henning, U. (1964) On the colinearity of gene structure and protein structure. Proc. Natl. Acad. Sci. U. S. A. 51, 266-272[Medline] [Order article via Infotrieve] |
22. |
Yanofsky, C.,
Drapeau, G. R.,
Guest, J. R.,
and Carlton, B. C.
(1967)
The complete amino acid sequence of the tryptophan synthetase A protein (![]() |
23. | Rothman, F. G. (1987) in Gene-protein relationships in Escherichia coli alkaline phosphatase: competition and luck in scientific research. In Phosphate Metabolism and Cellular Regulation in Microorganisms (Torriani-Gorini, A. , Rothman, F. G. , Silver, S. , Wright, A. , and Yagil, E., eds) , pp. 307-311, American Society for Microbiology, Washington, D. C. |
24. | Sarabhai, A. S., Stretton, A. O. W., Brenner, S., and Bolle, A. (1964) Co-linearity of the gene with the polypeptide chain. Nature 201, 13-17[Medline] [Order article via Infotrieve] |
25. | Crick, F. H. C., Barnett, L., Brenner, S., and Watts-Tobin, R. J. (1961) General nature of the genetic code for proteins. Nature 192, 1227-1232 |
26. | Yanofsky, C., Berger, H., and Brammar, W. J. (1969) In vivo studies on the genetic code. Proc. Int. Congr. Genet. 3, 155-165 |
27. | Guest, J. R., and Yanofsky, C. (1965) Amino acid replacements associated with reversion and recombination within a coding unit. J. Mol. Biol. 12, 793-804[Medline] [Order article via Infotrieve] |
28. |
Helinski, D. R.,
and Yanofsky, C.
(1963)
A genetic and biochemical analysis of second-site reversion.
J. Biol. Chem.
238,
1043-1048 |
29. |
Nagata, S.,
Hyde, C. C.,
and Miles, E. W.
(1989)
The ![]() |
30. | Brody, S., and Yanofsky, C. (1963) Suppressor gene alteration of protein primary structure. Proc. Natl. Acad. Sci. U. S. A. 50, 9-16[Medline] [Order article via Infotrieve] |
31. | Carbon, J., Berg, P., and Yanofsky, C. (1966) Missense suppression due to a genetically altered tRNA. Cold Spring Harbor Symp. Quant. Biol. 31, 487-497[Medline] [Order article via Infotrieve] |
32. | Gupta, N., and Khorana, H. G. (1966) Missense suppression of the tryptophan synthetase A protein mutant A78. Proc. Natl. Acad. Sci. U. S. A. 56, 772-779[Medline] [Order article via Infotrieve] |
33. | Berg, P. (1973) Suppression: a subversion of genetic decoding. Harvey Lect. 67, 247-272[Medline] [Order article via Infotrieve] |
34. | Goldberg, M. E., Creighton, T. E., Baldwin, R. L., and Yanofsky, C. (1966) Subunit structure of the tryptophan synthetase of Escherichia coli. J. Mol. Biol. 21, 71-82[Medline] [Order article via Infotrieve] |
35. | Pan, P., Woehl, E., and Dunn, M. F. (1997) Protein architecture, dynamics and allostery in tryptophan synthase channeling. Trends Biochem. Sci. 22, 22-27[CrossRef][Medline] [Order article via Infotrieve] |
36. | Cox, E. C., and Yanofsky, C. (1967) Altered base ratios in the DNA of an Escherichia coli mutator strain. Proc. Natl. Acad. Sci. U. S. A. 58, 1895-1902[Medline] [Order article via Infotrieve] |
37. | Franklin, N., Dove, W. F., and Yanofsky, C. (1965) The linear insertion of a prophage into the chromosome of E. coli shown by deletion mapping. Biochem. Biophys. Res. Commun. 18, 898-909 |
38. | Morse, D. E., Mosteller, R. D., and Yanofsky, C. (1969) Dynamics of synthesis, translation, and degradation of trp operon messenger RNA in E. coli. Cold Spring Harbor Symp. Quant. Biol. 34, 725-740[Medline] [Order article via Infotrieve] |
39. | Baker, R. F., and Yanofsky, C. (1968) Direction of in vivo degradation of a messenger RNA. Nature 219, 26-29[Medline] [Order article via Infotrieve] |
40. | Imamoto, F., Ito, J., and Yanofsky, C. (1966) Polarity in the tryptophan operon of E. coli. Cold Spring Harbor Symp. Quant. Biol. 31, 235-249[Medline] [Order article via Infotrieve] |
41. | Imamoto, F., and Yanofsky, C. (1967) Transcription of the tryptophan operon in polarity mutants of Escherichia coli. I. Characterization of the tryptophan messenger RNA of polar mutants. J. Mol. Biol. 28, 1-23[Medline] [Order article via Infotrieve] |
42. | Somerville, R. L., and Yanofsky, C. (1964) On the translation of the A gene region of tryptophan messenger RNA. J. Mol. Biol. 8, 616-619 |
43. | Yanofsky, C., Platt, T., Crawford, I. P., Nichols, B. P., and Christie, G. E. (1981) Nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res. 9, 6647-6668[Abstract] |
44. | Bauerle, R. H., and Margolin, P. (1967) Evidence for two sites for initiation of gene expression in the tryptophan operon of Salmonella typhimurium. J. Mol. Biol. 26, 423-436[Medline] [Order article via Infotrieve] |
45. | Jackson, E. N., and Yanofsky, C. (1972) Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene. J. Mol. Biol. 69, 307-313[Medline] [Order article via Infotrieve] |
46. | Horowitz, H., and Platt, T. (1982) Identification of trp-p2, an internal promoter in the tryptophan operon of Escherichia coli. J. Mol. Biol. 156, 257-267[Medline] [Order article via Infotrieve] |
47. |
Jackson, D. A.,
and Yanofsky, C.
(1969)
Restoration of enzymic activity by complementation in vitro between mutant ![]() ![]() |
48. | Kelley, R. L., and Yanofsky, C. (1985) Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. Proc. Natl. Acad. Sci. U. S. A. 82, 483-487[Abstract] |
49. | Bennett, G. N., and Yanofsky, C. (1978) Sequence analysis of operator constitutive mutants of the tryptophan operon of Escherichia coli. J. Mol. Biol. 121, 179-192[Medline] [Order article via Infotrieve] |
50. | Gunsalus, R. P., and Yanofsky, C. (1980) Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc. Natl. Acad. Sci. U. S. A. 77, 7117-7121[Abstract] |
51. | Rose, J. K., Squires, C. L., Yanofsky, C., Yang, H. L., and Zubay, G. (1973) Regulation of in vitro transcription of the tryptophan operon by purified RNA polymerase in the presence of partially purified repressor and tryptophan. Nat. New Biol. 245, 133-137[Medline] [Order article via Infotrieve] |
52. | Squires, C. L., Lee, F. D., and Yanofsky, C. (1975) Interaction of the trp repressor and RNA polymerase with the trp operon. J. Mol. Biol. 92, 93-111[Medline] [Order article via Infotrieve] |
53. | Otwinowski, Z., Schevitz, R. W., Zhang, R. G., Lawson, C. L., Joachimiak, A., Marmorstein, R. Q., Luisi, B. F., and Sigler, P. B. (1988) Crystal structure of trp repressor/operator complex at atomic resolution. Nature 335, 321-329[CrossRef][Medline] [Order article via Infotrieve] |
54. | Zhang, H., Zhao, D., Revington, M., Lee, W., Jia, X., Arrowsmith, C., and Jardetzky, O. (1994) The solution structures of the trp repressor-operator DNA complex. J. Mol. Biol. 238, 592-614[CrossRef][Medline] [Order article via Infotrieve] |
55. | Yanofsky, C., Kelley, R. L., and Horn, V. (1984) Repression is relieved before attenuation in the trp operon of Escherichia coli as tryptophan starvation becomes increasingly severe. J. Bacteriol. 158, 1018-1024[Medline] [Order article via Infotrieve] |
56. | Kumamoto, A. A., Miller, W. G., and Gunsalus, R. P. (1987) Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators. Genes Dev. 1, 556-564[Abstract] |
57. | Lawson, C. L., and Carey, J. (1993) Tandem binding in crystals of a trp repressor/operator half-site complex. Nature 366, 178-182[CrossRef][Medline] [Order article via Infotrieve] |
58. | Roth, J. R., Silbert, D. F., Fink, G. R., Voll, M. J., Anton, D., Hartman, P. E., and Ames, B. N. (1966) Transfer RNA and the control of the histidine operon. Cold Spring Harbor Symp. Quant. Biol. 31, 383-392[Medline] [Order article via Infotrieve] |
59. | Doolittle, W. F., and Yanofsky, C. (1968) Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J. Bacteriol. 95, 1283-1294[Medline] [Order article via Infotrieve] |
60. | Jackson, E. N., and Yanofsky, C. (1973) The region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function. J. Mol. Biol. 76, 89-101[Medline] [Order article via Infotrieve] |
61. | Kasai, T. (1974) Regulation of the expression of the histidine operon in Salmonella typhimurium. Nature 249, 523-527[Medline] [Order article via Infotrieve] |
62. | Yanofsky, C., and Soll, L. (1977) Mutations affecting tRNA Trp and its charging and their effect on regulation of transcription termination at the attenuator of the tryptophan operon. J. Mol. Biol. 113, 663-677[Medline] [Order article via Infotrieve] |
63. | Morse, D. E., and Morse, A. N. (1976) Dual-control of the tryptophan operon is mediated by both tryptophanyl-tRNA synthetase and the repressor. J. Mol. Biol. 103, 209-226[Medline] [Order article via Infotrieve] |
64. | Lee, F., Squires, C. L., Squires, C., and Yanofsky, C. (1976) Termination of transcription in vitro in the Escherichia coli tryptophan operon leader region. J. Mol. Biol. 103, 383-393[Medline] [Order article via Infotrieve] |
65. |
Yanofsky, C.
(2000)
Transcription attenuation: once viewed as a novel regulatory strategy.
J. Bacteriol.
182,
1-8 |
66. | Yanofsky, C. (1981) Attenuation in the control of expression of bacterial operons. Nature 289, 751-758[Medline] [Order article via Infotrieve] |
67. | Merino, E., and Yanofsky, C. (2002) in Regulation by termination-antitermination: a genomic approach. In Bacillus subtilis and Its Closest Relatives: from Genes to Cells (Sonenshein, A. L. , Hoch, J. A. , and Losick, R., eds) , pp. 323-336, American Society for Microbiology, Washington, D. C. |
68. | Henkin, T. M., and Yanofsky, C. (2002) Regulation by transcription attenuation in bacteria: how RNA provides instructions for transcription termination/antitermination decisions. Bioessays 24, 700-707[CrossRef][Medline] [Order article via Infotrieve] |
69. | Henkin, T. M. (2000) Transcription termination control in bacteria. Curr. Opin. Microbiol. 3, 149-153[CrossRef][Medline] [Order article via Infotrieve] |
70. | Winkler, W., Nahvi, A., and Breaker, R. R. (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature 419, 952-956[CrossRef][Medline] [Order article via Infotrieve] |
71. |
Grundy, F. J.,
Winkler, W. C.,
and Henkin, T. M.
(2002)
tRNA-mediated transcription antitermination in vitro: codon-anticodon pairing independent of the ribosome.
Proc. Natl. Acad. Sci. U. S. A.
99,
11121-11126 |
72. | Lovett, M. A., and Rogers, E. (1996) Ribosome regulation by the nascent peptide. Microbiol. Rev. 60, 366-385[Abstract] |
73. |
Landick, R.,
and Yanofsky, C.
(1984)
Stability of an RNA secondary structure affects in vitro transcription pausing in the trp operon leader region.
J. Biol. Chem.
259,
11550-11555 |
74. | Fisher, R. F., Das, A., Kolter, R., Winkler, M. E., and Yanofsky, C. (1985) Analysis of the requirements for transcription pausing in the tryptophan operon. J. Mol. Biol. 182, 397-409[Medline] [Order article via Infotrieve] |
75. | Winkler, M. E., and Yanofsky, C. (1981) Pausing of RNA polymerase during in vitro transcription of the tryptophan operon leader region. Biochemistry 20, 3738-3744[Medline] [Order article via Infotrieve] |
76. |
Fisher, R.,
and Yanofsky, C.
(1983)
A complementary DNA oligomer releases a transcription pause complex.
J. Biol. Chem.
258,
9208-9212 |
77. | Landick, R., Carey, J., and Yanofsky, C. (1985) Translation activates the paused transcription complex and restores transcription of the trp operon leader region. Proc. Natl. Acad. Sci. U. S. A. 82, 4663-4667[Abstract] |
78. |
Toulokhonov, I.,
Artsimovitch, I.,
and Landick, R.
(2001)
Allosteric control of RNA polymerase by a site that contacts nascent RNA hairpins.
Science
292,
730-733 |
79. |
Khodursky, A. B.,
Peter, B. J.,
Cozzarelli, N. R.,
Botstein, D.,
Brown, P. O.,
and Yanofsky, C.
(2000)
DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli.
Proc. Natl. Acad. Sci. U. S. A.
97,
12170-12175 |
80. | Hershfield, V., Boyer, H. W., Yanofsky, C., Lovett, M. A., and Helinski, D. R. (1974) Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U. S. A. 71, 3455-3459[Abstract] |
81. |
Oppenheim, D. S.,
and Yanofsky, C.
(1980)
Translational coupling during expression of the tryptophan operon of Escherichia coli.
Genetics
95,
785-795 |
82. | Das, A., and Yanofsky, C. (1989) Restoration of a translational stop-start overlap reinstates translational coupling in a mutant trpB-trpA gene pair of the Escherichia coli tryptophan operon. Nucleic Acids Res. 17, 9333-9340[Abstract] |
83. | Di Martino, P., Merieau, A., Phillips, R., Orange, N., and Hulen, C. (2002) Isolation of an Escherichia coli strain mutant unable to form biofilm on polystyrene and to adhere to human pneumocyte cells: involvement of tryptophanase. Can. J. Microbiol. 48, 132-137[CrossRef][Medline] [Order article via Infotrieve] |
84. |
Wang, D.,
Ding, X.,
and Rather, P. N.
(2001)
Indole can act as an extracellular signal in Escherichia coli.
J. Bacteriol.
183,
4210-4216 |
85. | Stewart, V., Landick, R., and Yanofsky, C. (1986) Rho-dependent transcription termination in the tryptophanase operon leader region of Escherichia coli K-12. J. Bacteriol. 166, 217-223[Medline] [Order article via Infotrieve] |
86. | Stewart, V., and Yanofsky, C. (1985) Evidence for transcription antitermination control of tryptophanase operon expression in Escherichia coli K-12. J. Bacteriol. 164, 731-740[Medline] [Order article via Infotrieve] |
87. |
Gong, F.,
and Yanofsky, C.
(2002)
Analysis of tryptophanase operon expression in vitro: accumulation of TnaC-peptidyl-tRNA in a release factor 2-depleted S-30 extract prevents Rho factor action, simulating induction.
J. Biol. Chem.
277,
17095-17100 |
88. |
Gong, F.,
and Yanofsky, C.
(2002)
Instruction of translating ribosome by nascent peptide.
Science
297,
1864-1867 |
89. | Yanofsky, C., Horn, V., and Gollnick, P. (1991) Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli. J. Bacteriol. 173, 6009-6017[Medline] [Order article via Infotrieve] |
90. | Schneider, W. P., Nichols, B. P., and Yanofsky, C. (1981) Procedure for producing hybrid genes and proteins and its use in assessing the significance of amino acid differences in homologous tryptophan synthetase polypeptides. Proc. Natl. Acad. Sci. U. S. A. 78, 2169-2173[Abstract] |
91. | Yanofsky, C. (1984) Comparison of regulatory and structural regions of genes of tryptophan metabolism. Mol. Biol. Evol. 1, 143-161[Abstract] |
92. | Henner, D., and Yanofsky, C. (1993) in Biosynthesis of aromatic amino acids. In Bacillus subtilis and Other Gram Positive Bacteria: Biochemistry, Physiology and Molecular Genetics (Losick, R., ed) , pp. 269-280, American Society for Microbiology, Washington, D. C. |
93. | Gollnick, P., Babitzke, P., Merino, E., and Yanofsky, C. (2002) in Aromatic amino acid metabolism in Bacillus subtilis. In Bacillus subtilis and Its Closest Relatives: from Genes to Cells (Sonenshein, A. L. , Hoch, J. A. , and Losick, R., eds) , pp. 233-244, American Society for Microbiology, Washington, D. C. |
94. | Antson, A. A., Otridge, J., Brzozowski, A. M., Dodson, E. J., Dodson, G. G., Wilson, K. S., Smith, T. M., Yang, M., Kurecki, T., and Gollnick, P. (1995) The structure of trp RNA-binding attenuation protein. Nature 374, 693-700[CrossRef][Medline] [Order article via Infotrieve] |
95. | Antson, A. A., Dodson, E. J., Dodson, G., Greaves, R. B., Chen, X., and Gollnick, P. (1999) Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA. Nature 401, 235-242[CrossRef][Medline] [Order article via Infotrieve] |
96. |
Du, H.,
and Babitzke, P.
(1998)
trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis.
J. Biol. Chem.
273,
20494-20503 |
97. |
Sarsero, J. P.,
Merino, E.,
and Yanofsky, C.
(2000)
A Bacillus subtilis operon containing genes of unknown function senses tRNA Trp charging and regulates expression of the genes of tryptophan biosynthesis.
Proc. Natl. Acad. Sci. U. S. A.
97,
2656-2661 |
98. |
Valbuzzi, A.,
and Yanofsky, C.
(2001)
Inhibition of the B. subtilis regulatory protein TRAP by the TRAP-inhibitory protein, AT.
Science
293,
2057-2059 |
99. |
Valbuzzi, A.,
Gollnick, P.,
Babitzke, P.,
and Yanofsky, C.
(2002)
The anti-trp RNA-binding attenuation protein (anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein.
J. Biol. Chem.
277,
10608-10613 |
100. |
Jurgens, C.,
Strom, A.,
Wegener, D.,
Hettwer, S.,
Wilmanns, M.,
and Sterner, R.
(2000)
Directed evolution of a (beta alpha) 8-barrel enzyme to catalyze related reactions in two different metabolic pathways.
Proc. Natl. Acad. Sci. U. S. A.
97,
9925-9930 |
101. | Yanofsky, C. (2001) Advancing our knowledge in biochemistry, genetics, and microbiology through studies on tryptophan metabolism. Annu. Rev. Biochem. 70, 1-37[CrossRef][Medline] [Order article via Infotrieve] |