Connecting a viral DNA replication apparatus with gene expression

seminars in VIROLOGY, Vol 6, 1995: pp 25-33

Connecting a viral DNA replication apparatus with gene expression E. Peter Geiduschek

core enzyme (E) by the small (185 amino acid) o family protein encoded by phage T4 gene 55 (gp55) (this is, to my knowledge, the original use of the abbreviation 'g-p', standing for gene product), and E.gp55 suffices for accurate and passably efficient initiation of transcription in vitro at T4 late promoters in negatively supercoiled DNA. However, viral DNA is not supercoiled in vivo (at least not on average)1 and the requirements for transcribing the phage T4 late genes in the infected E. colicell are more complex. (1) An additional small (112 amino acid) RNA polymerase-binding protein encoded by T4 gene 33 (gp33) is required. (2) Transcription of the late genes begins after the start of viral DNA replication and is conditional on DNA replication. If replication is interrupted, late transcription normally declines very substantially. Thus, full-scale late gene transcription is not just conferred by having the corresponding genes present in high copy number, but is more directly connected to DNA replication. (3) The coupling between phage DNA replication and late transcription can be broken under special circumstances that lead to the introduction of large numbers of breaks and gaps and into unreplicated DNA. 2"6 Bacteriophage T4 encodes an entire replication machinery: a DNA polymerase (gp43), its three accessory proteins, consisting of the gp44/62 complex (a DNA-dependent ATPase) and g'p45, a single stranded DNA-binding protein (gp32), DNA helicase (gp41) and primase (gp61), a protein that tightens the binding of the helicase-primase complex to DNA (gp59), and an additional DNA helicase (dda).7'a The gp44/62 complex and gp45 make DNA chain elongation by the T4 DNA polymerase very processive. 9.t° Homologues of these three proteins confer processivity on prokaryotic and eukaryotic replicating DNA chain elongation: for gp45, the weU-studied homologues are the dimeric ~ subassembly of the E. coli DNA polymerase III holoenzyme, and the proliferating cell nuclear antigen, PCNA, which is associated with eukaryotic nuclear DNA polymerase 8; the homologues of the gp44/62 complex (also DNAdependent ATPases) are the five-subunit y complex of

The mechanisms that generate the connection between bacteriophage 7"4 DNA replication and activation of the viral late genes are described. Transcriptional activation requires enhancer-like DNA sites that serve as loading points for the direct transcriptional activator gp45, (the T4 gene 45 protein). Gp45 also confers processivity on replicative DNA chain elongation. The structures of the g~O45-homologous replication proteins that track along DNA are discussed, and an interpretation of their principal modes of action is offered. A closing section presents some speculations about the prospect for recognizing the operation of similar mechanisms in other biological systems.

Key words: gene regulation / transcription replication-transcription coupling / enhancer DNA-tracking protein

/ /

MAre VXRUS~Smake the expression of some part of their genomes dependent on replication. The strategy, which is shared among large and small, RNA as well as DNA viruses, is doubtless capable of being realized through diverse mechanisms. In this article, specific emphasis is placed on the particular mechanism that generates a connection between DNA replication and transcription of the viral late genes in the development of bacteriophage T4. Recent biochemical work provides a reasonably detailed understanding of this mechanism of transcriptional regulation and brings to light unusual (in the sense of hitherto unrecognized) relationships. Approximately 40% of the ~ 165 kbp bacteriophage T4 genome is accessible from nearly 40 extremely simple promoters, each consisting of TATAAATA (or, in a few cases, variants of TATAAATA) centered approximately 10 bp upstream of a transcriptional start site. The ability to recognize these simple promoters is conferred on E. coli RNA polymerase From the Department of Biology and Centerfor Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA ©1995 AcademicPressLtd 1044-5773/95/010025 +0958.00/0

25

E.P. Geiduschek

appropriately adjusted to depress the basal activity of E.gp55, the d e p e n d e n c e of transcription on these replication proteins is almost absolute. Transcriptional activation requires the ATPase activity of these three proteins, and also requires the DNA nick, which has the formal properties of an enhancer, since it can be placed at a great distance from, and either upstream or downstream of, the T4 later protnoter. T h e fact that it is a structure rather than a sequence makes it an unusual kind of enhancer. Two other properties set it apart from conventional enhancers (which serve as specific binding sites for protein assemblies that activate transcription by interaction with initiation complexes through space): 12'13 (1)The T4 late p r o m o t e r and its e n h a n c e r must be connected by a continuous and clear path along DNA; DNA templates in which the three T4 DNA polymerase accessory proteins are unable to activate T4 late transcription include constructs (Figure 1) in which the e n h a n c e r and p r o m o t e r are topologically but not covalently linked, and constructs in which all paths

the E. coli DNA polymerase III holoenzyme, and the five-subunit replication factor (RF)-C. H Gp43, 44, 62 and 45 are together referred to as the phage T4 DNA polymerase holoenzyme.

The effect of replication proteins on T4 late transcription A simple in-vitro system that is satisfactory for analysing the involvement of replication proteins in T4 late transcription has been constructed. It is based on the use ofplasmids containing a T4 late transcription unit and a site at which a single strand break (nick), introduced into a specific DNA strand, serves as the potential starting point for DNA chain elongation by the T4 replication protein. Because transcription of this nicked DNA by the basal E.gp55 RNA polymerase is relatively inactive, stimulation by the three DNA polymerase accessory p r o t e i n s - - g p 4 4 , 62 and 4 5 - - i s readily detected. Indeed, u n d e r conditions that are

P23014)

(314)

P23(4201

(420)

~

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Figure 1. Design of a plasmid for analysing T4 late description in vitro, and for demonstrating the required connectivity of enhancers and promoters. The plasmid pDH72AE1 ( ~ 3900 bp; structure 1) contains two T4 late transcription units with identical promoters (P2a) that yield different size transcripts (314 and 420 nt, respectively). When pDH72AE1 is cut at the indicated site ('nick') in the non-transcribed strand of these two transcription units by the endonuclease encoded by the filamentous phage gene II, this creates the enhancer of transcription at both of the T4 late promoters. The nicking site is flanked by binding sites for EcoRI restriction endonuclease, E2 and E3. A mutant EcoRI protein (E--*Q at amino acid 111) that is nucleolytically defective binds very tightly to these sites, blocking the path between the enhancer and each of the two T4 late promoters. Plasmid pDH72AE1 also contains two recognition sites for the y6 resolvase, a sitespecific recombinase whose action yields the singly linked catenane shown at the top fight (structure II), in which the enhancer is covalently connected to one of the two T4 late transcription units and topologically, but not covalently linked to the other. The diagram at the far fight shows the spatial relationships between transcription units and enhancer in structure II, with the DNA nick represented by its 5'--*3' polarity. In structure I, activation of both of the T4 late transcription units by the three T4 DNA polymerase accessory proteins is blocked by binding EcoRI Gln 111 protein at E1 and E2. In structure II, the T4 DNA polymerase accessory proteins activate only the transcription unit that is covalently connected to the enhancer (adapted from ref 16). 26

Viral DNA replication apparatus along DNA between the enhancer and the promoter are blocked by protein. (2) Transcriptional enhancement is constrained with regard to polarity, in that the DNA nick that serves as the enhancer must be located in the non-transcribed strand of the target transcription unit. These special properties of the enhancer of T4 late transcription suggest that it is an entry or assembly site for one or more proteins and that transcriptional activation requires protein tracking between the enhancer and its promote~: 14-16 Enhancer-dependent transcriptional activation by the three DNA polymerase accessory proteins in vitro also requires the T4 gp33 as a co-activator, while u n e n h a n c e d basal transcription is further depressed by gp33.15 Thus, it appears that the specialized function of this small RNA polymerase-binding protein is to enforce the d e p e n d e n c e of late transcription in the phage T4-infected cell on activation by replication proteins. Further analysis establishes two distinct roles for the T4 DNA polymerase accessory proteins: gp45 is the direct activator of transcription and ultimately becomes stably associated with enhanced transcription initiation complexes at T4 late promoters. The gp44/62 complex serves as the assembly factor of gp45, is required for loading gp45 onto DNA, but has no further role in transcriptional activation. The current understanding of the enzymology of T4 DNA replication provides a plausible view of these roles: 7's'17 ATP binding promotes the attachment of gp44, 62 and 45 to primer-template junctions. ATP hydrolysis, the catalytic site for which resides in gp44, releases gp45 from such complexes for tracking along DNA. 17'1s A model explaining the mechanism of transcriptional activation in these terms is shown in Figure 2 ) 8 The DNA nick that serves as the transcriptional enhancer is the site of assembly of the g p 4 4 / 6 2 / 4 5 complex (Figure 2, lines a and b). The polarity of the nicked DNA strand determines the orientation of the asymmetric g p 4 4 / 6 2 / 4 5 complex, and particularly of gp45, on DNA. ATP hydrolysis releases gp45 to track freely along DNA, but the orientation that is imposed at loading is subsequently retained without inversion. As it tracks along DNA, gp45 encounters RNA polymerase and can bind firmly to it only if the polymerase also has gp33 and gp55 b o u n d to it. The orientation of gp45 determines the interaction-compatible orientation of RNA polymerase and therefore the polarity of the p r o m o t e r that can be transcriptionally enhanced (Figure 2, line c). The above model of the transcriptional activation is further supported by the observation that the obliga-

tory participation of the gp44/62 complex can be eliminated under special conditions (of 'macromolecular crowding'). Under these circumstances, transcriptional activation still requires gp45 and gp33, but is not ATP hydrolysis-dependent, does not require the DNA nick-as-enhancer and is not restricted with regard to polarity. 21 Thus, it is the gp44/62 assembly factor and the local structure of DNA that determine the orientation of gp45.

The structures of replication proteins that track along DNA, and an interpretation of their principal modes of action The mechanism of transcriptional activation that has just been laid out derives visual immediacy from recently determined protein structures. As already pointed out, the E. coil ~ protein, PCNA and gp45 are functional homologues) 1 E. coli [3 has been identified as a dimeric protein that is constrained to track along DNA and the five-protein E. coli DNA polymerase I I I y complex has been identified as an ATP hydrolysisrequiring assembly factor for ~.22 The structure of the [3 dimer is remarkable: 2s it is a ring with an approximately 35 2~ diameter central hole, amply large enough for a DNA double helix, and lined on the DNA-proximal inner surface with positively charged amino acid side chains. The two lateral faces of the dimer are non-identical. Each [3 m o n o m e r has three structurally similar domains of nearly equal size, so that the ~2 ring has a pseudo six-fold symmetry (Figure 3). When the [32 structure was presented, it was suggested that trimeric gp45 and PCNA would prove to have similar structures and this conjecture has been borne out, in a general sense, in the very recently determined structure of PCNA. ~4 No more vividly and intuitively compatible structure for DNA-tracking proteins could be imagined. It is simple to see how the catenated [3 dimer would confer processivity on DNA polymerase, tethering it to DNA by direct p r o t e i n protein interaction, without constraining it to any fixed site. That the threading of such a processivity factor onto DNA might require the assistance of an assembly factor is also readily conceivable. It is necessary to point out that some of this is conjectural for T4 gp45, since its structure has not yet been determined. However, there is direct evidence for gp45 tracking along DNA in the absence of DNA polymerase or RNA polymerase 2s and gp45 does 27

E.P. Geiduschek

of promoter-bound RNA polymerase, the catenated protein might facilitate the DNA twisting that accompanies strand separation in the xdcinity of the ta'anscriptional start site. In contrast, there is no reason to expect an important role for such a protein in RIgA chain elongation, because the intrinsic processivity of E. coli RNA polymerase core enzyme is very high indeed.

interact directly with gp43, the T4 DNA polymerase.26 In the absence of other attributes, what can a DNA-tethered DNA polymerase processivity factor contribute to transcriptional activation? (1) If it can make DNA scanning by RNA polymerase more processive, such a protein might cut down the time required to find promote~-s. (2) By tethering one end

PROMOTER

a.

NICK

5'

3I 5'

3'

b.

5'

dADP + Pi + gp44/62

f

C,

......

5~

NOTENHANCED

ENHANCED

Figure 2. A model of T4 late promoter activation by the DNA polymerase accessory proteins. (a) The nick in DNA serves as the binding site for the assembly factors, gp44/62, and as the entry site for the transcription-enhancing protein, gp45. (b) When gp45 is loaded onto DNA, its orientation is determined by the orientation of the gp44/62 complex at the nick. (c) ATP hydrolysis dissociates gp45 from the gp44/62-DNA complex, releasing gp45 to track freely across the DNA. The orientation of gp45 is solely compatible with activation of RNA polymerase in one orientation (e.g. with RNA polymerase at promoter P.yThe representation of the RNA polymerase core (E., hatched in gray) is modeled after the structure of bacteriophage T7 RNA polymerase 19 and scanning transmission electronmicrographs ofE. coliRNA polymerase core, 2° with respect to its organization around a large cleft capable of accommodating a DNA helix, much as the palm of a hand grips a rod. Only RNA polymerase core enzyme that bears gp55 can recognize the promoter and only enzyme bearing gp33 interacts productively with gp45. However, the precise contact points between gp45 and the RNA polymerase-gp55-gp33 complex remain to be determined (reproduced from ref 18, with permission). 28

Viral DNA replication apparatus The connection between phage T4 DNA r e p l i c a t i o n a n d late t r a n s c r i p t i o n

transcription, but does n o t explain why replication itseff s h o u l d be required, or how the strong quantitative effect o f o n g o i n g D N A r e p l i c a t i o n on late transcription d u r i n g p h a g e T4 infection is genei'ated. In-vitro experiments have provided s o m e g u i d a n c e by

T h e p r e c e d i n g discussion rationalizes the involvem e n t o f certain replication proteins in p h a g e T4 late

Figure 3. Representation of the structure of E. coli 6. The two-fold axis of this dimer runs through the center of the structure, and through the printed page. Each monomeric subunit has three structurally similar domains. Thus, the entire structure has a pseudo six-fold symmetry, whereas the sides of the ring facing the reader and facing away from the reader are chemically disdnct and capable of distinctive protein-protein interactions. The diameter of the central hole is - 35 ,/t, not taking tightly bound water into account, with ample room for the DNA helix that is shown (modelled, and not part of the crystallographicaily solved structure) on the axis. I am grateful to J. Kuriyan, Rockefeller University, for this black and white version of illustrations previously presented in color in ref 23. 29

E.P Geiduschek

showing that concurrent DNA replication has only modest quantitative effects on transcriptional activation, and by suggesting that this quantitative effect is generated by converting DNA nicks into more preferred loading sites for gp45. 27 It is a current conjecture that DNA replication continuously generates enhancers o f T 4 late transcription (at one time the replisome and replication fork were referred to as the 'mobile enhmacer' of T4 late transcription; ref 14). Gp45 is expected to be part of the replisome but discontinuous lagging strand DNA replication (Figure 4) constantly creates short-lived potential gp45 loading sites (they are short-rived because discontinuous DNA fragments are rapidly j o i n e d together). In

the space between two divergent replication forks, these enhancers allow both polarities of gp45 loading and generate transcriptional activation that is globally unrestricted with regard to polarity. If the persistence of the tracking-competent state of gp45 is limited (we imagine that competence to track along DNA is conferred by the loading of gp45 onto DNA as a protein catenane (Figure 3) that is not entirely stable and tends to dissociate), then the tracking population of gp45 must be continuously replenished. If DNA replication is interrupted, new discontinuous DNA fragments are not created and those remaining are rapidly joining together. If the consequent depletion of enhancers depresses the rate of gp45 assembly onto

Figttre 4. Divergent replication forks and replisomes (lightly shaded ovals) generate a space within which discontinuous (lagging strand) DNA replication has generated sites for loading gp45 ,~shaded .and filled double rings) onto DNA in each of the two possible orientations. Gp45 can traak:£rom its ,entry sites, .as shown, to target T4 late transcription units that are also oriented in either direction. 30

Viral DNA replication apparatus DNA, this must be gradually reflected in a depletion of tracking protein, and a diminished rate of formation of gp45-activated T4 late promoter complexes. In this plausible though hypothetical mechanism, we see that the coupling of T4 late transcription to concurrent replication is not an intrinsic and inexorable aspect of transcriptional activation by a DNAtracking protein, but arises from a particular set of quantitative characteristics: the limited lifetime of the DNA-tracking state of this particular transcriptional activator, gp45, and the continuous turnover of its DNA-loading sites in a cell.

mal chromatin. It might also be argued that the kind of mechanism that connects T4 viral DNA replication to late gene expression must be peculiarly viral, suitable to systems undergoing many DNA doublings within a relatively short time and producing replication proteins at a high rate. That is a plausible argument, but not an exclusion principle. Moreover PCNA, for example, is not an especially rare protein. 3~,s4 The phenotype of T4 late transcription, namely its nearly absolute d e p e n d e n c e on DNA replication and strong quantitative coupling to concurrent DNA replication, should not be regarded as an obligatory signature of this kind of transcriptional activation mechanism, as already pointed out. Replicationcoupled transcriptional activation is probably generated through specific secondary features, such as the instability of DNA-loading sites for gp45, and the limited persistence of the DNA tracking-competent state of gp45. A quantitatively different set of these specific parameters might, in some other system, yield a qualitatively different phenotypic signature. There is a close parallel between T4 late gene regulation and the properties of herpes simplex virus HSV-1 class Y2 genes, whose transcription is abolished if DNA replication is blocked by phosphonoacetic acid. aS'a6 Some, but not all, y~ promoters have relatively simple structures. 37'38 However, a mutant in the HSV-1 gene coding for its replication-associated single stranded DNA-binding protein makes class Y2 RNA in the absence of substantial viral DNA syntheses. s9 It has also been shown that certain class "/9 genes are u n d e r the transcriptional control of site-specific DNA-binding transcription factors. 40 Moreover, Y2 class genes are not quantitatively uniform, that is, not entirely coordinate in their response to DNA replication. Thus, the phenotypic parallels are less impressive than they might at one time have appeared to be. 41 With the more complete understanding of the relevant mechanism that is now at hand, one realizes, however, that the above list of properties of HSV-1 Y2 genes is indecisive in regard to molecular mechanism of the transcriptional activation. Given the fact that the lifetimes of DNA-tracking catenanes and of their special assembly sites on DNA can, in principle, vary over a wide range, that the DNA-tracking transcriptional activators may function in combination with transcriptional effectors that bind to fixed, specificsequence DNA sites, that the states of transcriptional activation that are generated can, at least in eukaryotes, have appreciably long lifetimes, the phenotypic characteristic linking c o n c u r r e n t DNA replication

Where might the operation o f similar mechanisms be encountered? In a separate article in this issue, Rothman-Denes discusses the role of the phage N4 single-stranded DNA-binding protein in phage DNA replication, recombination and transcription. Omission of a detailed and direct comparison between T4 and N4 late gene expression reflects our j u d g m e n t that different fundamental mechanisms are involved. However, it does seem intrinsically likely that the general mechanism that generates activation of bacteriophage T4 late promoters by a DNA-tracking replication protein is utilized in other, natural biological systems. DNA replication, recombination and probably also repair 11'28-31 load protein catenanes onto DNA at distinctive sites, such as discontinuities of the prima@ structure. I anticipate that the properties that make gp45 and its functional homologues suitable for DNA replication also make them too generally suitable for transcriptional regulation for that potentiality to have been exploited in only a single instance. Where might one look in order to discover further instances of the operation of such mechanisms? It might be argued that the mechanism that has been described here must be specialized to prokaryotes and to those lower eukaryotes that do not have nucleosomal chromatin, because nucleosomal arrays and chromatin superstructures must severely restrict the range of DNA-tracking proteins, s2 This argument is unpersuasive on several counts: (1) the loading and dispersion of DNA-tracking protein can be established during a time interval in which replication sweeps the chromatin forming proteins off DNA; (2) when a DNA-tracking protein enhances the formation of stable transcriptional preinifiafion complexes, that generates a potentially persistent gene activation; (3) some animal viral DNA is not organized as nucleoso31

E.P. Geiduschek 5. Riva S, Cascino A, Geiduschek EP (1970) Uncoupling of late transcription from DNA replication in bacteriophage T4 development. J Mol Biol 54:103-119 6. Karam, J.D. (1994) Molecular biology of bacteriophage T4. American Society for Microbiology, Washington, DC 7. Alberts BM (1987) Prokaryotic DNA replication mechanisms, Philos Trans R Soc London Ser B 317:395-420 8. Nossal N (1994) The DNA replication fork, in Molecular Biology of Bacteriophage T4 (KaramJD, ed. in chief), pp 43-53. American Society for Microbiology 9. Huang CC, Hearst JG, Alberts BM (1981) Two types of replication proteins increase the rate at which T4 DNA polymerase traverses the helical region in a single-stranded DNA template. J Biol Chem 254:4087-4094 10. Reddy MK, Weitzel SE, yon Hippel PH (1993) Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase. Proc Nati Acad Sci USA 90:3211-3215 11. Kuriyan J, O'Donnell M (1993) Sliding clamps of DNA polymerases. J Mol Biol 234:915-925 12. Ptashne M, Gann AA (1990) Activators and targets. Nature 346:329-331 13. Su W, Porter S, Kustu S, Echols H (1990) DNA-looping and enhancer activity: association between DNA-bound NtrC activator and RNA polymerase at the bacterial GlnA promoter. Proc Nail Acad Sci USA 87:5504-5508 14. Herendeen DR, Kassavetis CA, Barry J, Alberts BM, Geiduschek EP (1989) Enhancement of bacteriophage T4 late transcription by components of the T4 DNA replication apparatus. Science 245:952-958 15. Herendeen DR, Williams KP, Kassavetis CA, Geiduschek EP (1990) An RNA polymerase-binding protein that is required for communication between an enhancer and a promoter. Science 248:573-578 16. Herendeen DR, Kassavetis CA, Geiduschek EP (1992) A transcriptional enhancer whose function imposes a requirement that proteins track along DNA. Science 256:1298-1303 17. Gogol EP, Young MC, Kubasek WL, Jarvis TC, yon Hippel PH (1992) Cryoelectron microscopic visualization of functional subassemblies of the bacteriophage T4 DNA replication complex. J Mol Biol 224:395-412 18. Tinker RL, Williams KP, Kassaveds CA, Geiduschek EP (1994) Transcriptional activation by a DNA-tracldng protein: structural consequences of enhancement at the T4 late promoter. Cell 77:225-237 19. Sousa R, Chung Yj, Rose JP, Wang BC (1993) Crystal structure of bacteriophage T7 RNA polyrnerase at 3.3 /~ resolution. Nature 364:593-599 20. Tichelaar W, Van Heel M (1990) Characteristic views of Escherichia co//RNA polymerase core enzyme in the scanning transmission electron microscope, j Struct Biol 103:180-184 21. Sanders GM, Kassavetis CA, Geiduschek EP (1994) Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gp45 and late transcription. Proc Natl Acad Sci USA 91:7703-7707 22. Stukenberg PT, Studwell VP, O'Dormell M (1991) Mechanism of the sliding beta-clamp of DNA polymerase III holoenzyme. J Biol Chem 266:11328-11334 23. Kong XP, Onrust R, O'Donnell M, Kuriyan J (1992) Threedimensional structure of the beta subunit of E. coli DNA polymerase HI holoenzyme: a sliding DNA clamp. Cell 69:425-437 24. Krishna TSR, Kong X-P, Gary S, Burgers PM, KuriyanJ (1994) Ca'ystal structure of the eukaryotic DNA polymerase processivity factor PGNA. Cell 79:1233-1243 25. Tinker RL, Kassavetis CA, Geiduschek EP (1994) Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA. EMBO J 13:5330-5337

with gene activation cannot be taken as a complete or secure way of identifying candidates for this class of gene regulatory mechanism. It appears at this time that direct biochemical analysis may provide the most secure path to further progress in identifying the range of application of this class of transcriptional activators. In regard to HSV-1, progress in understanding the enzymo!ogy of its DNA replication 42'43 encourages the anticipation that a direct test of these speculations may soon be feasible. In closing, it is worth pointing to a special relationship between the DNA-tracking catenanes and sitespecific DNA-binding proteins. Each class of protein has the ability to recruit diverse effectors to the vicinity of DNA by protein-protein interaction. The DNA-binding proteins bring their ligands to fixed sites, whereas the catenanes confer their own onedimensional mobility on their ligands. 44 The corresponding possibilities for gene regulation must differ in interesting ways (one might consider the different consequences, for example, of constraining DNA to a cellular matrix by means of a site-specific DNAbinding protein and a proteinaceous retaining ring).

Acknowledgements I am grateful to colleagues and collaborators who have contributed the experimental findings and many of the ideas that constitute the foundations of this essay, in particular, D.1L H e r e n d e e n , G,~. Kassavetis, G. Sanders, P~L. Tinker, ICE Williams, a n d J . Winkelman. I also thankJ. Kuriyan (Rockefeller University) for Figure 3. O u r research on this subject has been supported by grants from the National Institute o f General Medical Sciences.

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