Regulated recruitment and cooperativity in the design of biological regulatory systems

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1 / rsta Regulated recruitment and cooperativity in the design of biological regulatory systems By M a r k Ptashne Memorial Sloan{Kettering Cancer Center, 1275 York Avenue, Box 595, New York, NY 10021, USA (m-ptashne@ski.mskcc.org) Published online 6 May 2003 What distinguishes a man from a mouse is not so much di erent proteins, but rather the appearance of common proteins (etc.) at di erent times and positions in the developing organisms. Thus speci c genes are transcribed or repressed, proteins degraded or stabilized, RNA transcripts spliced one way or another, and so on. These are examples of `regulatory decisions. A rather simple mechanism called regulated recruitment lies at the heart of many of these regulatory decisions. Keywords: regulated recruitment; cooperative binding; evolvability; synergy; enzyme speci city; gene activation 1. Introduction A goal of this symposium was to identify simple rules, or strategies, that can be used to develop complex systems. I describe here a simple molecular strategy that nature has used widely in evolving biological complexity. This description is part of an extended argument put forth in the recently published book Genes and signals (Ptashne & Gann 2002). The biological complexity I refer to has two aspects, one having to do with the development of a complex organism from a fertilized egg, and the other with the evolution of di erent forms of life. A similar theme is sounded in the two cases: limited sets of gene products (proteins, mainly and RNA molecules) are used, in di erent combinations, to generate diversity. For example, proteins that generate human hands also generate human feet, but the di erent appendages form as a result of di erent patterns of appearance of these proteins as the embryo develops. And, speaking somewhat more informally, a common set of gene products can be used to generate a human, a mouse, and perhaps even a y, by appropriately regulating their appearances as each of these organisms develops. There is more to it than this, of course, but these statements su ce to pose the following problems. How is the appearance of proteins regulated during development of any one organism? What kinds of molecular changes to the regulatory apparatusare required to One contribution of 18 to a Theme `Self-organization: the quest for the origin and evolution of structure. 361, 1223{ c 2003 The Royal Society

2 1224 M. Ptashne Figure 1. Cooperative binding. Interactions between proteins binding to DNA (for example) are typically weak, involving, say, 1{2 kcal of interaction energy. The resulting e ect on binding, say 10- to 100-fold, can be physiologically important. Modi ed with permission from Ptashne & Gann (2002). modify those patterns as evolution proceeds? To return to our original theme, how is biological complexity generated from a limited set of common elements? 2. Protein regulation There are many ways to regulate the appearance of a protein in a cell at a given time and place, but for the purposes of illustration we consider just three: a gene can be transcribed (or not); a RNA molecule can be spliced, sometimes one way or another; and a protein can be destroyed (by proteolysis). In each case the regulation can be either/or, or it can be graded: a gene can be transcribed more or less, and so on. My general point is that a common molecular strategy has been used in the evolution of regulatory systems that e ect each of these three kinds of control (and many others as well). The mechanism is called `regulated recruitment. To understand it we need rst to consider the ubiquitous phenomenon of cooperative binding, as illustrated in gure 1. I will then illustrate how the principle is used in transcription, discuss some of the implications, and then return, near the end, to RNA splicing and proteolysis. Figure 1 shows three macromolecules; we will call A and B `proteins, and the rod `DNA, but the following characteristics apply more generally. Given speci ed a nities of A and B for their sites, and assuming that A and B are present at approximately some speci ed concentrations (as would be found in a cell, for example), neither A nor B is bound e ciently to its site when the other protein is absent. When both proteins are present, however, both proteins are bound, an e ect called cooperative binding of proteins to DNA. The word `cooperativity is laden with historical associations, and so we have to be clear what is required in this case:

3 Regulated recruitment and cooperativity 1225 Figure 2. Gene activation e ected by cooperative binding. The rod is a DNA fragment containing the lacz gene, a site that binds the activator CAP, and a more extended sequence that binds the RNA polymerase, called the promoter. The `activator CAP binds DNA only when complexed with the small molecule cyclic AMP. Glucose in the medium depletes intracellular cyamp, and so CAP only works when glucose is absent from the medium. Reproduced with permission from Ptashne & Gann (2002). the proteins must simply `touch one another when bound to DNA. Conformational changes, though they may occur, are not necessary for the e ect, nor is energy in the form of adenosine triphosphate (ATP) (for example) used. Rather, one protein helps the other bind simply by providing binding energy in the form of an `adhesive interaction between the proteins. To see how the reaction of gure 1 applies to gene regulation, consider gure 2, which shows a bacterial transcriptional activator in action. The gene is the famous lacz (beta-galactosidase) gene of Escherichia coli. (For reasons that become evident upon considering the entire argument of Ptashne & Gann (2002), analysis of gene activation, rather than gene repression the usual entry into the subject of gene regulation is the more revealing approach). The catabolite activator protein (CAP), binds speci cally to its site on DNA, and RNA polymerase binds to its site, called the promoter. In the absence of CAP (and also in the absence of another regulatory protein, the lac repressor, see below), RNA polymerase binds to the promoter, but only infrequently. CAP binds cooperatively with the bacterial RNA polymerase as shown, and thereby `activates the gene. Note that the term `activation as used here can be misleading. Neither the enzyme nor the gene has in any meaningful sense been `activated ; rather, the activator merely increases the frequency (by cooperative binding) with which the constitutively active polymerase encounters the gene. We say the activator `recruits the polymerase to the gene. How do we know that the mechanism of gure 2 accurately describes activation of the lac and other bacterial genes? And how do we know that simple recruitment can account for activation of a typical eukaryotic gene? The latter question seems particularly di cult in view of the fact that over 65 proteins are required to transcribe a typical eukaryotic gene, and these proteins are found to be distributed in various complexes in the cell (see gure 4). Part of the proof is as follows. The mechanism of gure 2 makes an array of experimental predictions, all of which have been veri ed for certain genes in bacteria. Figure 2 implies the following.

4 1226 M. Ptashne Figure 3. An activator by-pass experiment in E. coli. This experiment has been successfully performed with di erent interacting pairs of proteins. In each case one is fused to a DNA-binding domain, the other to polymerase. In the case depicted here, a carboxyl terminal domain of the largest subunit of polymerase has been replaced by protein X. Reproduced with permission from Ptashne & Gann (2002). (i) The enzyme (RNA polymerase) is not pre-bound to the gene prior to activation. (ii) DNA binding of the activator is absolutely required for gene activation. Even when expressed at high concentrations, an activating region that is not tethered to DNA cannot `activate (because it cannot recruit). (iii) The detailed mode of DNA binding is irrelevant. That is, many di erent detailed modes of DNA binding of the activator will su ce to position it so that it can contact RNA polymerase. (iv) Two well-separated interaction surfaces are found on each protein. Thus, the polymerase has a DNA (promoter)-binding surface and, in addition, a site that contacts the activator. The activator has one site that binds DNA and another, called its `activating region, that contacts polymerase. (v) A unique activating region{enzyme interaction is not required. That is, any of an array of positions on the polymerase can be contacted by an activator to e ect cooperative binding and gene activation. (vi) Activator-bypass experiments work. An example of such an experiment is shown in gure 3. In place of an ordinary activator we have a fusion protein bearing a DNA-binding domain and an attached protein called Y. The polymerase has been modi ed so as to bear protein X, and X and Y are known to interact. Strong transcription ensues: a result showing that even a totally heterologous protein{protein interaction su ces for gene activation (provided one of the interacting components is tethered to DNA). The argument is given particular power thanks to the fact that there are two modes of gene activation found in bacteria that are di erent from the simple reaction shown here. Each of the diagnostic predictions just listed does not hold for one or the other or both of these alternative forms of gene activation in bacteria. (The bacterial genes activated by alternative mechanisms include a few involved in resistance to heavy metals and a group involved in nitrogen metabolism. For an explanation of how activation works in these cases see Ptashne & Gann (2002).)

5 Regulated recruitment and cooperativity 1227 Snf2 Med6 Sin4 TFIIF RNAPII TFIIB Srb4 Srb6 Srb10 TFIIE Kin28 CTD TFIIH TAFII145 TFIID SAGA Figure 4. Gene activation in eukaryotes. The typical transcriptional activator (green) is shown bound to DNA: it comprises a DNA-binding domain fused to a peptide that works as an activating region. The activating region recruits (and assembles) the large complex required to transcribe a gene. Many components of the transcriptional machinery are pre-assembled into subcomplexes (SAGA, TFIIH, etc.), as shown in blue. A segment of DNA is shown wrapped around histones (red) to form a nucleosome. 3. Activation in yeast All of these diagnostic experimental predictions have also been realized in yeast, some in a particularly striking fashion. For example, the yeast activator Gal4 can be split into two parts, neither of which activates on its own: one binds DNA and the other bears an activating region. The activating region will work when attached to a heterologous DNA-binding domain, even a bacterial binding domain (e.g. that of the E. coli repressor protein LexA), provided the test gene bears the appropriate DNA-binding site. The activating region itself resembles `glue or Velcro R : any of a wide array of sequences (particularly sequences containing an excess of hydrophobic and acidic residues) will work, and certain of those sequences have been shown to work with an e ciency approximately proportional to their lengths. The `two hybrid system, widely used in yeast to discover interacting protein pairs, depends on the facts that activating regions must be tethered to DNA to work, and that the mode of attachment to DNA is irrelevant. One modi cation required as we move from bacteria to yeast is that, in the latter case, the activator evidently does not contact the polymerase itself. Rather, it is believed, other components of the complex machinery of gure 4, some of which themselves bind polymerase, are recruited directly by the activator.

6 1228 M. Ptashne Figure 5. Hypothetical steps in evolution of regulation of the lac genes. In the top line, there is no regulation, and the polymerase spontaneously binds and transcribes the gene at a low level. In the second line a CAP site has been introduced upstream of the promoter, and CAP activates as in gure 1. In the third line, a lac-repressor binding site, called an operator, has been introduced. Now the gene can be transcribed only if lactose is present, because a metabolic product of lactose binds to repressor and prevents it from binding DNA. Having made the jump from bacteria to yeast, it is easy to see that many genes in higher eukaryotes must also be activated by recruitment. Thus, for example, Gal4 and other yeast activators can work in a wide array of higher organisms. Moreover, the overall nature of the transcriptional machinery, comprising the many proteins required for transcription of the typical gene, is essentially conserved between yeast and higher eukaryotes. It is therefore not surprising that (to take just one example) no higher eukaryotic gene, to my knowledge, can be activated by expressing, or even over-expressing, an activator that cannot bind DNA and hence cannot recruit. How a eukaryotic activator assembles the large complex required for transcription is currently under investigation. It is likely that some of the components are directly touched and recruited by the activator, others then binding cooperatively with the directly recruited components. The question now arises as to why evolution exploited regulated recruitment so widely for gene regulation. What might be features of that mechanism that make it particularly `evolvable (a term the meaning of which becomes clearer as we discuss the matter). What are the advantages over the other mechanisms found in bacteria that we have alluded to but not discussed explicitly here? Once again, I will not discuss those alternative mechanisms here, but rather will make a few general remarks about the evolvability of regulated recruitment. 4. Genetic regulation It is easy to see how, in principle, nature can start with a simple system and then, in steps, make it ever more sophisticated. Figure 5 shows the imagined evolution of regulation of the lac genes of E. coli. Starting with just the gene and the polymerase

7 Regulated recruitment and cooperativity 1229 Figure 6. Synergistic activation. Two activators that do not interact with each other are shown simultaneously binding to and recruiting polymerse. The two activators work synergistically : the level of transcription elicited by the two activators working together is more than the sum of the two activators working separately. (that is, in the absence of any regulation) the gene is transcribed at a low but biologically signi cant level. The bacterium constitutively synthesizes beta-galactosidase and can use lactose as a carbon source. The rst modi cation introduced is the activator, CAP, which, as we have seen, binds cooperatively with polymerase and thereby `activates transcription. The interaction of CAP with polymerase is small in terms of interaction energy (some 1{2 kcal) but that interaction increases transcription signi cantly, some 10- to 100-fold. The second modi cation is the addition of the repressor: this protein, by binding to a site within the promoter, excludes binding of polymerase, and its e ect overrides the action of CAP. These two additions turn an unregulated system into a rather sophisticated one: the gene is on if and only if lactose is present in the medium, and it is transcribed fully only if glucose (a better carbon source than lactose) is absent (see captions to gures 2 and 5 for more details). The system is also highly evolvable in another way. Because the speci city of CAP action depends only upon the position of its DNA-binding site (i.e. which gene it is near), it would seem quite straightforward to add that site to other genes. Indeed some 200 genes in E. coli are activated by CAP. Moving the CAP site from one gene to another, or adding it to new genes, e ects a change, or an extension, of the `meaning of a signal. CAP responds to glucose in the medium (see gure 2) and so changing where CAP acts (i.e. at which gene) means changing the meaning of the signal (which is glucose in this case). The system is highly modular. This modularity is found at many levels (including at the structure of the activator). For many genes and activators, any activator will work on any gene, precisely because what activators do is so simple. This mechanism recruiting a common enzyme to whatever gene one wishes to express should be contrasted to a hypothetical world in which a new enzymatic machinery would be built for each gene (see Ptashne & Gann 1998). Building complex organisms from a rather `small set of genes requires signal integration: genes must be regulated in response to combinations of regulators. For example, a given gene might be activated only when worked on by several activators. Regulated recruitment lends itself to signal integration in an obvious way. Thus, any two (for example) activators, binding near a gene will work synergistically, because

8 1230 M. Ptashne c-jun ATF-2 IRF-3 IRF-7 IRF-3 NFkB c-jun ATF-2 IRF-3 IRF-7 IRF-3 NFkB Figure 7. The human interferon-beta enhancer. Three activators (called Jun/ATF, IRF3/7 and NfkB) are shown binding cooperatively (with small auxiliary proteins) to form an `enhancesome. The enhancesome forms, and the gene is activated, only when all three activators are present and capable of binding DNA. Modi ed with permission from Ptashne & Gann (1998). each will contribute to the binding reaction as shown in gure 6. This e ect is easily observed in yeast: in general, any two activators placed near a gene work synergistically, even if those activators are unrelated and do not interact directly with each other. As we move to higher eukaryotes we have ever more signal integration in regulating genes. Figure 7 shows an extension of the ideas we have discussed: in this case three separate activators bind cooperatively to form an `enhancesome that activates the human interferon-beta gene. Only if all three activators are working does the gene become activated. These (and many other) eukaryotic activators are found working in di erent combinations with di erent partners. This `combinatorial control expands even further the ways that signals can be integrated. 5. Lambda switch The interactions I have been discussing between enzyme, activator and DNA, and between di erent activators as they bind DNA are instantiations of the cooperative binding mechanism illustrated in gure 1. The simple `adhesive interactions required for cooperative binding can be used to generate an `on{o switch that responds in an all-or-nothing manner to an extra-cellular switch. The familiar example, the bacteriophage lambda switch, is shown in gure 8. The lambda repressor (despite its name) activates transcription of its own gene as it represses transcription of an adjacent gene. Repressor monomers are in concentration-dependent equilibrium with dimers, as shown, and those dimers bind cooperatively to two adjacent sites on DNA. This autocatalytic system depends (speaking roughly) on the fourth power

9 Regulated recruitment and cooperativity 1231 RNA polymerase repressor gene Or3 Or2 Or1 lytic genes Prm Pr repressor gene RNA polymerase ON Or3 Or2 Or1 lytic genes OFF Prm Pr Figure 8. Autoregulation of the lambda repressor gene. The small blue dumb-bell-shaped protein is the repressor. It forms dimers, two of which bind cooperatively to sites (called Or1 and Or2) in the repressor gene s promoter, as shown. The repressor recruits the polymerase and activates its own gene (at promoter Prm) as it excludes polymerase from the adjacent promoter (Pr). The start sites and directions of transcription of the regulated genes are indicated by the yellow arrows. At higher concentrations repressor negatively regulates transcription of its own gene by binding to the third site shown (Or3) (see text). Other features of the lambda system reinforce the on{o nature of the switch (see Ptashne 1992; Ptashne & Gann 2002). of the monomer concentration. An inducing signal (e.g. UV light), which destroys repressor, has little e ect until the repressor concentration is reduced to a critical point, and from thereon induction occurs essentially instantaneously. Each of the reactions depicted in the gure between repressor monomers, between dimers and between repressor and polymerase is merely adhesive. And so a rather sophisticated switch has been produced by molecules that interact like Lego r Toys. But there is another reason I bring up the lambda switch. I wish to illustrate a very general problem that arises from the use of cooperativity (regulated recruitment) to control genes. Going back to our basic description of gure 1, we see that, for the binding of proteins A and B to be dependent upon one another, their concentrations must be below a speci ed level. The typical interaction between cooperatively binding proteins is weak: some 1{2 kcal, as we have already noted for a particular case (see lac genes above). That means that the system is highly sensitive to the concentrations of the interacting proteins. In the lambda case, for example, if the repressor concentration rises (say 5- to 10-fold) above the level found in cells, the cooperative e ects on repressor binding to DNA are lost. For example, the binding

10 1232 M. Ptashne Figure 9. Control of splicing by regulated recruitment. The SR protein works analogously to the transcriptional activator of gure 1: it recruits an enzyme, in this case the splicing machinery, to a potential splice site in the RNA. Di erent SR proteins, working with various cooperatively binding partners, bind speci c splicing enhancer sites in RNA and thereby promote one or another splicing event. Modi ed with permission from Graveley et al. (1999). of one dimer no longer depends on binding of the other. And so the `fourth power dependence of the switch, and hence its on{o character, would be lost. Lambda has evolved a seemingly elaborate mechanism for controlling the level of repressor in the cell. The principle is simple: at higher concentrations, the repressor binds to a DNA site called Or3 (see gure 8) and turns o its own synthesis. To e ect this negative feedback, the repressor binds cooperatively not only to sites in and around its promoter, but simultaneously with sites located some 3 kb (3000 base pairs) away. A large looped DNA structure is thereby formed, the sole purpose of which is to maintain the concentration of repressor below a speci ed level in the cell. All systems controlled by regulated recruitment must keep the concentration of key components below speci ed levels, and nature has found many ways to do this. For example, eukaryotic transcriptional activators are typically held outside the nucleus unless they are signalled to work, thus keeping their concentrations in the nucleus low. Some of the bewildering complexities of regulatory systems re ect nature s various ways of controlling the concentrations of active forms of various regulators. Now I can return to the two other examples I mentioned at the start: RNA splicing and proteolysis. Recall that regulation of these two processes, like regulation of transcription, can determine when, where, and to what extent any given protein might appear in a developing embryo. Figure 9 shows that a large splicing machine (comprising some 145 proteins) is recruited to a speci c site on RNA, where it then works spontaneously. Just as with RNA polymerase in E. coli and the more complex polymerase and associated proteins in eukaryotes, the enzyme depicted here works spontaneously at a low level on many di erent splice sites. Only when recruited to a speci c site does it work with high e ciency. Figure 10 shows how regulated recruitment controls proteolysis. A large protein complex adds ubiquitin molecules to a protein destined to be destroyed by another large complex called the proteosome. The gure illustrates how a protein is chosen to be ubiquitylated: an adaptor (or `activator ) protein called an F-box protein binds cooperatively with the target protein and with the ubiquitylating complex, and the ubiquitination reaction then proceeds spontaneously.

11 Regulated recruitment and cooperativity 1233 Figure 10. Control of ubiquitylation by regulated recruitment. The F-box protein brings a speci c substrate to the ubiquitylating enzyme. If the substrate speci city of the F-box protein is altered so that it recognizes a di erent protein, that protein will then be recruited and ubiquitylated. 6. Conclusion I will end with a slightly di erent formulation of what I have stated before. We might recast the problem as one of enzyme speci city. The classical enzymes we all studied many years ago (e.g. beta-galactosidase) have highly speci c active sites that typically recognize one and only one substrate. The term `regulation as applied to these enzymes (where it applies) usually refers to allosteric inhibitory or activating e ects of substrates and other small molecules. But many of the enzymes studied by molecular biologists, including those we have discussed here, can work on many di erent substrates: E. coli RNA polymerase can transcribe any of 3000 genes, for example; the ubiquitinating machinery can work on thousands of proteins in the cell; and so on. The regulatory problem thus reduces to one of substrate choice: which gene will be transcribed, which protein will be ubiquitylated, which RNA site will be spliced, and so on. When viewed in this way we see that nature has hit upon a generally applicable trick for imparting speci city: the use of compact protein domains, which can be inserted at many places on the surfaces of proteins, and which impart speci city for binding to another macromolecule. We have, for example, families of DNA-binding domains (e.g. helix{turn{helix and zinc- nger motifs) that direct transcriptional regulatory proteins to one or another site on DNA; families of protein binding domains (e.g. Sh2 and Sh3) that direct one protein to another; other domains that direct proteins to speci c sites on RNA molecules or on membranes; and so on. The speci city of each domain is limited, and we usually nd two or more of these locator domains working cooperatively. `New genes that are found in higher eukaryotes often encode the same enzyme found in a lower eukaryote but attached to arrays of `locator domains that give the enzyme a new speci city in the sense I have been discussing. The general principle extends even further than this brief description: `locator functions can be provided by molecules other than proteins. For example, in the process called RNA interference, a bit of double-stranded RNA recruits, to a speci c mrna site, a machinery that destroys that mrna. RNA works as a recruiter in

12 1234 M. Ptashne several other instances as well (directing telomerase to speci c sequences at the ends of chromosomes, for example), and other examples are likely to be discovered. I have stressed, by way of illustration, how enzymes, sometimes found in large complexes, can be directed to one or another substrate by regulated recruitment, and have only mentioned in passing that such regulatory events often occur in response to extra-cellular signals. A more complete discussion would reveal that the pathways which convey signals to the regulatory apparatus, so-called `signal transduction pathways, are themselves made up of essentially common enzymes (e.g. protein kinases) that are given speci city by the kinds of simple binding interactions I have been discussing (see Ptashne & Gann 2002). Pawson (2003) describes in more detail the plethora of `locator domains found in nature and how they are used in various ways to create these signal transduction pathways. References Graveley, B. R., Hertel, K. J. & Maniatis, T SR proteins are `locators of the RNA splicing machinery. Curr. Biol. 9, R6{R7. Pawson, T Organization of cell-regulatory systems through modular-protein-interaction domains. Phil. Trans. R. Soc. Lond. A 361, 1251{1262. Ptashne, M A genetic switch: phage lambda and higher organisms, 2nd edn. Oxford: Blackwell Science. Ptashne, M. & Gann, A Imposing speci city by localization: mechanism and evolvability. Curr. Biol. 8, 812{822. Ptashne, M. & Gann, A Genes and signals. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.