The Chemistry of Regulation of Genes and Other Things (2014)

The truth of a theory lies in the deductive methods used to establish it and the experimental demonstration of its fundamental premises and consequences—Jacques Monod I had the good fortune, early on, to be gripped by a scientific problem, gene regulation, that had ramifications beyond what I imagined. Its unfoldings have kept me enthralled ever since. We began with bacteria, and especially with bacteriophage λ, and then moved to work with yeast and mammalian cells. We always sought coherent descriptions, ideas that would apply to apparently disparate cases, regulation of prokaryotic and eukaryotic genes, for example, despite the fact that the latter, but not the former, are sequestered in a nucleus and wrapped in nucleosomes. My goal here is to put the various stages of our understanding, from the beginning of my involvement, in an overarching context. I might not say anything that has not been said by others or me, but my hope is that otherwise obscure connections and simplifications will be made clear. I hope I avoid distortions, but I cannot cite all the important contributions of others. More detailed explanations for various experiments and arguments, along with more extensive references, can be found in my two books (1.Ptashne M. A Genetic Switch: Phage Lambda Revisited. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2004Google Scholar, 2.Ptashne M. Gann A. Genes & Signals. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2002Google Scholar) and in the references here. Where I refer to “we,” I of course mean to include the crucial role of this or that student or postdoctoral fellow. They are identified in the reference list but not in the text. I spent the summer of 1961 working in the laboratory of Aaron Novick and Frank Stahl at the University of Oregon, down the road from where I was finishing my undergraduate degree at Reed College in Portland, Oregon. I was sent to that lab by Ed Novitski, the master Drosophila geneticist, after a blissful summer of fly work at the Rocky Mountain Biological Laboratory in Crested Butte, Colorado. Major personalities appeared in Crested Butte: Ed himself, of course, H. J. Muller, Bruce Baker, Charles Remington, and so on, all in serious good humor. Ed's motives in sending me to Aaron and Frank are suspect: he despised the rumblings of molecular biology, the end of intelligent science. Perhaps he thought I would mix up the tubes and bring the whole enterprise to a halt. I did in fact mix up the tubes, but the effect was more of a hiccup than a demolition. Frank, who I later learned was in a transient tiff with his pal Matt Meselson, took the clue from Ed and told me there was only one person under whom I should do my Ph.D.: Matt. Thus, the next year, I followed Matt's move, orchestrated by Jim Watson, from Caltech to Harvard (the college, not the medical school), where I entered graduate school. Only at Harvard did I realize what a serious place Reed had been. I should have noticed: it seemed that half the students entering Reed, and often the most interesting ones, dropped out. There was a limit, apparently, to which serious self-indulgence (on the one hand) and serious work (on the other) successfully mixed. At Reed, you actually had to be able to discuss the two-hundred or so pages of reading that you had been assigned. At Harvard, the key question usually worked: will that be on the exam? In Oregon, I had encountered dazzling ideas emanating from the Institut Pasteur in Paris. François Jacob, Jacques Monod, and colleagues, working with bacteria, had proposed the existence of regulatory molecules called “repressors” that would turn off expression of specific genes unless inactivated by specific extracellular signals. The word “specific” requires attention, as it will come up often in this essay. Here, it signifies that one repressor, the λ phage repressor, was proposed to maintain most of the forty or so genes of the virus in a dormant state (i.e. “off”) in a lysogenic bacterium. And a different repressor, the Lac repressor, was proposed to control expression of the lac genes, genes required for metabolism of the sugar lactose. The signal that inactivated each repressor was specific, too: UV light in the λ case and lactose (or a metabolic derivative thereof) in the Lac case. Inactivation of the λ repressor, it was proposed, switched on expression of dormant phage genes and thereby initiated a lytic cycle of phage growth; in the Lac case, the signal (lactose) elicited expression of the lac genes only when needed (i.e. in the presence of lactose). I recently have touched upon our indebtedness to the French scientists in presenting this picture to us in my reflections on Jacob (3.Ptashne M. François Jacob (1920–2013).Cell. 2013; 153: 1180-1182Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). We were inspired by the dream, a bit vague at the time, that the repressor (what it is, what it does, and how) would illuminate development of a complex organism from a fertilized egg. Even then, we surmised that formation of different body parts requires differential expression of common genes and that different organisms can develop using essentially the same set of genes. λ was especially interesting because it presented us with the “memory” problem: once lysogeny was established in a bacterium, that state of gene expression was perpetuated for very many generations in the absence of an inducing signal. Neither “remembering” nor switching required any mutation. The switch is thus “epigenetic”: I had not thought the matter through when I incorrectly called it a “genetic” switch (1.Ptashne M. A Genetic Switch: Phage Lambda Revisited. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2004Google Scholar). Analogous switches had to underlie intermediate and final stages of development, we thought. However, work on the repressor had to wait. As Frank put it, I needed a license (i.e. a Ph.D. degree) to do experimental science on my own. So I went to work, learning the ropes, on a tangential problem involving the growth of phage λ. Amazingly, by the time I had completed work for my Ph.D. degree in Matt's lab in 1965 and despite rumors of serious efforts, no one had managed to get their hands on one of these putative repressors. I say “amazingly” because four years is a long time in this field. We were thereby given a gift in the form of a difficult but, as it turned out, not unsolvable task. As a junior fellow of the Society of Fellows at Harvard (from 1965 to 1968), not only did I get a free dinner every week, but I also was given my own lab, right next door to E. O. Wilson, the great ant man himself. (I think Wilson took a dim view of our operation; he had not planned to study the behavior of ants labeled at random with 32P.) So we began the assault. At that point, the repressor was defined as the product of a phage gene (called c1) based on the effects of mutations in that gene. The repressor had no known enzymatic activity and was inferred to be present in low amounts, perhaps as few as 10–100 molecules/cell. Nowadays, of course, one would overexpress the protein in cells using recombinant DNA techniques, and isolating the gene product would not be a serious challenge. We used the opposite approach to raise the relative amount of repressor synthesis in cells: we in effect destroyed (or nearly so) all bacterial and phage genes except c1 and fed the damaged cells radioactive amino acids. We detected a radiolabeled protein that was absent or had changed properties if the c1 gene was deleted or damaged (4.Ptashne M. Isolation of the λ phage repressor.Proc. Natl. Acad. Sci. U.S.A. 1967; 57: 306-313Crossref PubMed Google Scholar). Bingo! “We” here is meant to include Nancy Hopkins, whose name, consistent with the blinkered custom of the times, does not appear on the first few λ repressor papers. She was, at the time and before becoming a graduate student, a technician who made crucial contributions to the work. Was the product of the c1 gene actually a repressor, or might it have been, for example, an enzyme that converted some other molecule into the real repressor (an unlikely but possible scenario)? Could the c1 gene product on its own regulate a gene, or was it part of some more complicated apparatus? Did the repressor work directly on the DNA, the favored view, or at some other stage of gene expression? Specificity was the key. We mixed radiolabeled repressor with λ DNA, sedimented the mixture in a velocity gradient, and saw (with great excitement) that some of the repressor was bound to the fast-sedimenting DNA. (Nowadays gel electrophoresis or, so-called “chromatin immuno-precipitation,” and not centrifugation, would be used for such an experiment. It saves a lot of space and money.) This binding was specific in the following sense. We knew, thanks to the French scientists, that a phage closely related to λ, called 434, made its own repressor that had no effect on λ gene expression, and, correspondingly, the λ repressor had no effect on 434. We found, first, that the λ repressor did not bind to 434 DNA under conditions in which it bound λ DNA (5.Ptashne M. Specific binding of the lambda phage repressor to lambda DNA.Nature. 1967; 214: 232-234Crossref PubMed Scopus (135) Google Scholar). Then, even better, we showed that the 434 repressor bound to 434 DNA but not to λ DNA (6.Pirrotta V. Ptashne M. Isolation of the 434 phage repressor.Nature. 1969; 222: 541-544Crossref PubMed Scopus (36) Google Scholar). The demonstration that the two repressors, λ and 434, bound with opposite specificities to the two different DNA molecules, consistent with their behaviors in vivo, left little room for doubt: these proteins could bind to specific DNA sites (called “operators”), and a surmise later confirmed (7.Steinberg R.A. Ptashne M. In vitro repression of RNA synthesis by purified lambda phage repressor.Nat. New Biol. 1971; 230: 76-80Crossref PubMed Scopus (33) Google Scholar), they prevent transcription of target genes. These experiments illustrate the power of working with pairs and of combining genetics and biochemistry, lessons I learned, among many others, from Matt. This description omits the drama: in an enormously stimulating competition, Wally Gilbert and his postdoctoral fellow Benno Müller-Hill raced us to be the first to “solve the repressor,” as Jim Watson would say. Let us call it a tie: they were marginally faster in detecting their repressor, the Lac repressor (8.Gilbert W. Müller-Hill B. Isolation of the Lac repressor.Proc. Natl. Acad. Sci. U.S.A. 1966; 56: 1891-1898Crossref PubMed Google Scholar, 9.Gilbert W. Müller-Hill B. The lac operator is DNA.Proc. Natl. Acad. Sci. U.S.A. 1967; 58: 2415-2421Crossref PubMed Scopus (192) Google Scholar), and we were, by a respectable margin, the first to show specific DNA binding of a repressor. The experiments demonstrating specific DNA binding did not reveal how this binding was achieved. Might unusual DNA sequences be involved? Might the DNA have to unwind to expose base pairs or form some unusual structure such as Z-DNA, a popular item back then? Had the repressor turned out to be an RNA molecule or to be attached to one, the problem would have seemed simpler. However, the repressor was a pure protein, and a series of indirect experiments soon indicated that it recognizes its affined specific DNA sequence in the standard helical form (10.Maniatis T. Ptashne M. Multiple repressor binding at the operators in bacteriophage λ.Proc. Natl. Acad. Sci. U.S.A. 1973; 70: 1531-1535Crossref PubMed Scopus (98) Google Scholar). This left us with a mystery. By now I had moved, spiritually if not physically, from junior fellow to lecturer at Harvard. There was some good bureaucratic reason for the odd title, but I cannot recall it now. Along with the rare postdoctoral fellow, a few students joined up, some of whom would not leave until they had done something interesting. Fortunately, no one had yet invented the “four (or even five) years and you are out” rule. We were not very specialized back then. With only a modest-sized lab, we performed genetic, physiological, and biophysical experiments with everyone thinking about everyone else's experiments. Our department included Matt Meselson, Jim Wang, Jim Watson, Steve Harrison, Don Wiley, Paul Doty, Wally Gilbert (newly arrived from physics), Guido Guidotti, Nancy Kleckner, Konrad Bloch, Jack Strominger, and, later (after finishing his postdoctoral research with me), Tom Maniatis. Group meetings were open, and the criticisms and brainstorming could be fierce, a kind of scientific paradise. X-ray crystallography and model building (first of a related protein, discussed below, called Cro) (11.Anderson W.F. Ohlendorf D.H. Takeda Y. Matthews B.W. Structure of the cro repressor from bacteriophage λ and its interaction with DNA.Nature. 1981; 290: 754-758Crossref PubMed Scopus (400) Google Scholar, 12.Pabo C.O. Lewis M. The operator-binding domain of λ repressor: structure and DNA recognition.Nature. 1982; 298: 443-447Crossref PubMed Scopus (390) Google Scholar, 13.Aggarwal A.K. Rodgers D.W. Drottar M. Ptashne M. Harrison S.C. Recognition of a DNA operator by the repressor of phage 434: a view at high resolution.Science. 1988; 242: 899-907Crossref PubMed Scopus (434) Google Scholar) suggested that an α-helix (we called it the “recognition helix”) could insert into the groove of ordinary B-form DNA. Amino acid functional groups extending from the helix would make specific contacts with edges of base pairs, we thought. A slew of structural and genetic experiments, including the crystal structure of the 434 repressor with its operator, confirmed this idea. For example, in a helix-swap experiment, we replaced the putative recognition helix on one repressor with that found on another and thereby changed the specificity of binding as predicted (14.Wharton R.P. Ptashne M. Changing the binding specificity of a repressor by redesigning an α-helix.Nature. 1985; 316: 601-605Crossref PubMed Scopus (128) Google Scholar). Our experiments on and thinking about structural problems were very much influenced by conversations and collaborations with Steve Harrison and Jim Wang at Harvard. An imaginative bit of combined sequence/structural analysis, performed by three former lab members in collaboration with R. F. Doolittle at the University of California, San Diego, pointed to a few critical residues that were present at a characteristic spacing in several known and putative DNA-binding proteins. This pattern, they correctly surmised, signaled the presence of a common HTH motif that presented the recognition helix for DNA binding (15.Sauer R.T. Yocum R.R. Doolittle R.F. Lewis M. Pabo C.O. Homology among DNA-binding proteins suggests use of a conserved super-secondary structure.Nature. 1982; 298: 447-451Crossref PubMed Scopus (287) Google Scholar). This soon led to the identification of HTH motifs in homeodomain proteins, factors crucial for development of higher eukaryotes (16.Laughon A. Scott M.P. Sequence of a Drosophila segmentation gene: protein structure homology with DNA-binding proteins.Nature. 1984; 310: 25-31Crossref PubMed Scopus (393) Google Scholar). However, these structural insights only partially solved our problem. Specificity is a matter of degree. Put roughly (pretty much the best we can do even now), this means that, at the concentration of repressor found in a lysogen, the repressor would be found bound much more frequently at the operator than at other places in the genome. The information read by a single recognition α-helix is obviously not sufficient to achieve this degree of selectivity; too many sequences identical to the five or so base pairs recognized by that α-helix would be strewn about by chance. In addition, even if the site were unique, the repressor would spend so much time on the sea of related sites that it would never find the right one (see Appendix 1 in Ref. 1.Ptashne M. A Genetic Switch: Phage Lambda Revisited. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2004Google Scholar). Two protein-protein interactions, in addition to the protein-DNA interactions, turned out to be critical as follows. In the first of these protein-protein interactions, repressor dimers form in with the higher the the higher the is present as This of repressor behavior was by velocity of the repressor, which by then we could V. R. Ptashne M. The λ and 434 phage Spring Biol. Google Scholar). This made because protein dimers are the binding site would be thereby DNA contacts with recognition Only where two of the recognized sequence to each other and with would the protein bind it that each repressor recognizes a sequence that is if not that I will not indicated that the of DNA binding was, first, formation of a (or of a higher and, binding to DNA V. Ptashne M. form of two PubMed Scopus Google Scholar). We soon learned, that even the relative of a site did not By I was a Harvard I had a with the at the he me he was this despite the of who my to this I in a put a to the Harvard that it to the In the and I think the actually but with little effect on Harvard or the we set about isolating a λ operator by λ DNA with in the presence of pure repressor. We that the repressor would and thus from a of The was that the repressor in addition to the other as (10.Maniatis T. Ptashne M. Multiple repressor binding at the operators in bacteriophage λ.Proc. Natl. Acad. Sci. U.S.A. 1973; 70: 1531-1535Crossref PubMed Scopus (98) Google Scholar). I soon on a scientific as I and to to with Tom a postdoctoral fellow in my to the sequences of some of these DNA early methods were with the that but they We found, to our that the operator (called for operator there is also an not one but, apparently, three The sites are but not identical and each is We labeled these sites and T. Ptashne M. Structure of the λ 1973; PubMed Scopus Google Scholar, T. Ptashne M. A. R. Recognition sequences of repressor and in the operators of bacteriophage Full Text PDF PubMed Scopus Google Scholar, T. Ptashne M. J. Sequence of a site in the DNA of bacteriophage PubMed Scopus Google Scholar). This was the only after into graduate that I (i.e. Tom in a laboratory other than my own. that, as the sites read left to right These especially and were it turned out, in various λ (i.e. that the repressor and in its Ptashne M. of The Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Scholar). three repressor sites in the not of the lies in the called repressor binding or, We the effect back at Harvard, we performed a of the called us to repressor the sites in as a of repressor Two operator sites and were first, and the site was only at higher repressor However, experiments with DNA single DNA each of the three sites from its showed that sites and had identical for the repressor, an some than that of site the presence of the of but not that of for the repressor Ptashne M. regulation by the λ phage repressor.Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). In other on DNA, the repressor to sites 1 and We surmised that the concentration of repressor in a is such that and are usually by repressor. The is a matter I will to The of the of sites in for the repressor is when the sites are and when the sites are on DNA binding of the repressor to DNA, we is to the case of binding of to However, in the case, the effect of one molecule on the binding of another is by a in the The four sites are and is inferred from the of the for as a of In our case, we could one repressor molecule binding to the another repressor bind to the does a repressor binding to site another bind to as discussed that the repressor has little or no effect on DNA so the of that would structural along the DNA to in upon seemed We that was by contacts DNA-binding repressors. This view was confirmed by two different of experiments, one and the other In collaboration with at University and using his we found that the repressor two as the was that the protein of two that can This was confirmed by the that two were by of the repressor that the and the other the and by the that the at two different as predicted C.O. R.T. Ptashne M. The lambda repressor two Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). The most important of this series of experiments, in the was that the domain the HTH and bound to the three sites in but did so the of binding of the domain to the three sites in their as Ptashne M. regulation by the λ phage repressor.Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar, R.T. Pabo C.O. Ptashne M. of the λ repressor in the PubMed Scopus Google Scholar). These indicated that the be by the This surmise was later confirmed by the of of the repressor that had the residues on a of the The interaction of repressor dimers is in or one or two amino acid but is sufficient to the of some A. Specificity for the interaction of λ repressor and repressor PubMed Scopus Google Scholar, A. Amino acid contacts at the of the bacteriophage λ and PubMed Scopus Google Scholar, mutations in the domain of bacteriophage λ repressor dimers bound to PubMed Scopus Google Scholar). We also learned, early on, that sites not to each other for the sites are a DNA which the proteins, the For example, a repressor binding to a single site we found, another bind to a site some The DNA that were under the As the sites were ever more closely to each only by of of the DNA helix (i.e. were bound A. Ptashne M. binding of λ to sites by of the DNA Full Text PDF PubMed Scopus Google Scholar, J. A. Ptashne M. DNA by binding of λ repressor.Nature. PubMed Scopus Google Scholar). This was as if the repressor DNA in essentially its B-form and if DNA has such that bound on opposite of the helix cannot each We called this a of the experiment. At the time, all of this was as it was or that the of DNA was such that would be the and very unlikely thanks to However, the was thanks to others, we encountered a repressor sites by on the λ (see The protein-protein do not have to be to be The of the interaction at λ is an in repressor concentration an in concentration of some the Thus, at even the repressor will to bind sites the effect that that at from the opposite of view, we that specificity of binding at of repressor than otherwise would be that are bound do not have to bind the same protein at of Where proteins bind we have a to only when (or all of proteins are present and able to bind DNA to a would any bind sites can be often especially in higher the binding proteins in be to the of DNA These are in later as we discuss the of transcription We next these does the repressor and how does it do The might get here. The λ repressor was its Lac called a repressor because genetic experiments first had its role in expression of lytic phage genes. However, we now that the protein as an and as a repressor, as discussed Genetic experiments of others indicated that the repressor expression