Mark Ptashne begins his “Reflections” article in The Journal of Biological Chemistry with a quote by French biochemist and Nobel laureate Jacques Monod: “(T)he truth of a theory lies in the deductive methods used to establish it and the experimental demonstration of its fundamental premises and consequences.”
Mark Ptashne begins his “Reflections” article in the Journal of Biological Chemistry with a quote by French biochemist and Nobel laureate Jacques Monod: “(T)he truth of a theory lies in the deductive methods used to establish it and the experimental demonstration of its fundamental premises and consequences.”
Ptashne’s article reviews and puts into context many of his experiments on the molecular basis of gene regulation, beginning with bacteria and then moving to yeast and mammalian cells. He emphasizes how, at each stage, alternative answers to problems were confronted and how key experiments distinguished between them. He also emphasizes that the principles at work in bacteria apply as well in eukaryotes.
While an undergraduate at Reed College in Portland, Ore., in the 1960s, Ptashne first learned of “dazzling ideas” emerging from the Institute Pasteur in Paris. There, Monod and Francois Jacob, studying gene regulation in bacteria, proposed that regulatory molecules called “repressors,” Ptashne explains, “turn off expression of specific genes unless inactivated by specific extracellular signals.”
Ptashne wanted to know: Was this idea correct? What were “repressors,” and how did they work?
In 1962, Ptashne entered graduate school at Harvard University. He writes that he and his colleagues were inspired by the dream that understanding “the repressor” would “illuminate development of a complex organism from a fertilized egg.” By then it was strongly suspected 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.” And so regulation was the key.
The behavior of the bacteriophage lambda, as pointed out by the French scientists, represented a paradigm. In a lysogenic bacterium, one repressor (the bacteriophage lambda repressor) keeps most of the virus’ nearly 40 genes in a dormant state (off). UV irradiation of these lysogens switches the regulatory program so that the silent genes are now on, and a new crop of phage is produced.
Lambda was particularly interesting to Ptashne and his colleagues because it exemplified the so-called “memory” problem. Once lysogeny was generated in a bacterium, he writes, “that state of gene expression was perpetuated for very many generations in the absence of an inducing signal. Neither ‘remembering’ nor switching requires any mutation.”
Ptashne completed his Ph.D. in 1965, became a junior fellow of the Society of Fellows at Harvard from 1965 to 1968 and started his own lab. There, he and his colleagues isolated the repressor, a pure protein, not an RNA molecule and not attached to one. They showed that this protein could bind to specific DNA sites (operators) on DNA and later showed that it could prevent transcription of target genes. Ptashne notes that the experiments showing that repressor binds DNA specifically and many other experiments performed along the way demonstrate the power of combining genetics and biochemistry.
By 1971, Ptashne was a professor. His lab’s next step was to determine how the repressor binds DNA. Their experiments, along with those of others, indicated that an α helix (the so-called recognition helix) at the repressor could insert into the major groove of B-form DNA. Amino acid functional groups extending from the helix would make specific contacts with base pairs. But even repressor dimers, which recognize sites of two-fold rotational symmetry, did not bind with sufficient specificity. Ptashne’s lab learned that cooperativity is essential; one protein (e.g., a repressor dimer) helps another dimer bind DNA by merely touching it. This simple binding reaction, it turned out, also explains transcription activation by a specific DNA-binding protein: The “activator” contacts RNA polymerase and helps it bind and work at a promoter that lies near the activator binding site.
|Lambda repressor and Cro action at the right operator (Or) in a lysogen and following induction (bottom line).
As Ptashne explains, a DNA-bound activator recruits polymerase to a promoter. The simplicity of the activation mechanism means any gene can be brought under the control of any activator by apposing the activator and polymerase binding sites. And a suitably positioned DNA-bound protein (e.g., lambda repressor) can turn off, or repress, certain genes as it activates others.
The solution to the lambda memory problem demonstrates that where an activator works on its own gene, that state of gene expression tends to self-perpetuate. As cells divide, the activator distributes to daughters, with the state maintained. Memory is thus a property of the system of basic elements appropriately arranged. “By combining these elements, nature can produce sophisticated switches, which allows genes to be expressed in alternate states, with sensitive and dramatic transitions between them in response to signals,” he writes.
Over the following years, Ptashne and colleagues described lambda’s switch as a complex set of interactions that guaranteed expression of alternate sets of genes with a rapid switch upon command.
The switch includes positive and negative feedback; a double negative circuit involving the repressor and the protein Cro; and cooperativity of repressor binding, including the example (demonstrated later by others) of interactions between proteins separated by 3,000 base pairs, with formation of a large DNA loop. A separate gene regulatory circuit establishes repressor synthesis in the first place.
As Ptashne notes, only rather simple binding interactions are required to construct such a switch, and it is not hard to imagine how it might have evolved. These aspects of systems biology are widely found in gene regulatory circuits driving development of higher organisms.
Lambda “remains the best-understood integrated system we have, and perhaps one should ponder how we got it.” He writes, “The switch was not deduced from general observations or theoretical or mathematical models. Its parts were assembled as we went along, its glorious integrated working revealed only near the end. At every stage we could test this or that aspect, challenging with genetics and biochemistry, trying to ensure that each bit was well in hand before going on.” He implies that today this is a rare undertaking: “This approach is nowadays rather out of fashion. Instead we have the ‘big picture,’ many genes, obscure words and formulations.”
Ptashne then turned his investigation to how the insights from the lambda work might apply to eukaryotes. He and his colleagues chose to work with yeast, a eukaryote that could be genetically manipulated almost as easily as bacteria. He writes that they “had no way of knowing, at the start, that studying λ repressor and its action would yield a coherent picture of a regulatory switch and even less indication that the principles of protein-DNA interaction and gene regulation, gleaned from the λ studies, would apply even in eukaryotes.”
They showed, however, that eukaryotic activators (e.g., the yeast protein Gal4) work, as does lambda repressor, as an activator – by recruitment. Only simple binding interactions are required, and it is thus easy to see how “natural variation can throw up many regulatory-circuit options for natural selection to consider.”
The fact that eukaryotic genes are wrapped in nucleosomes, whereas bacterial genes are not, presents a special problem for transcriptional activators, and Ptashne recounts his group’s recent experiments that indicate the way this problem is solved as well.
He observes that today diagrams of gene regulatory circuits look like “a lot of lambdas” and that regulatory proteins, usually working cooperatively and in many different combinations, turn on sets of genes. Some of these genes encode inhibitors that block the effects of activators. Once positive feedback loops are established, they maintain states of gene expression unless they are perturbed, such as by a signal, thus permitting the system to move on to the next phase of gene expression. He speculates that the key is evolution: “(S)election must occur in small steps, and the simplest mechanism that works will be used over and over again, sometimes in so many guises that the underlying similarities are at first hard to see.”
Ptashne ends his “Reflections” by noting that “one can make ever broader generalizations by solving basic problems, sometimes in near fanatical detail, and then seeing where those solutions can lead,” instead of “looking at problems in general.” In addition, he writes, “No part of the world can simply be read – it always must be interpreted, and those interpretations are subject to constant reevaluation.”