- Email: [email protected]
Transcription factors vs nucleosomes: regulation of the PH05 promoter in yeast
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T~BS 22 - MARCH 1997
Tran c plon adors vs aac eosomes: reg laloa of the PHO$ # omote yeast John Svaren arid Wolfram HSrz Activation of the Saccharomyces cerevisiae PH05 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chmmatin transition requires a DNA-binding protein, Pho4, and its tmnsactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosemes to PH05 regulation are reviewed.
ONE OF THE main challenges in the area of transcriptional regulation is to discover how signalling pathways impinge on the activity of DNA-binding proteins to alter patterns of gene expression. A success story in this regard has been the PHO system, which is involved in regulating phosphate metabolism in the cell and is poised to scavenge extracellular phosphate when internal pools become depleted. Based on the pioneering genetic studies of Oslima and coleagues, the PHO system has provided fettle ground for investigations of sigual transduction, transcriptional regulation and the function of chromatin structure. M0du[at0rs of PHO tmnscipt[0n Phosphate starvation o| the yeast Saccharomyces cerevisiae results in at least a 50-fold increased production of secreted acid phosphatase, which is composed of three isozymes produced from the PH05, PHOIO and PHOII genes t2. More than 90% of the acid phosphatase activity is supplied by the PH05 gene product, and experiments have therefore concentrated on its regulation. Positive modulators of PH05 transcription include Pho4 (Ref. 3), a basic helix-loophelix (bHLH) transactivator4, and Pho2 (Ref. 5), a homeobox DNA-binding protein6. Pho4 binds to two upstream activating sequence (UAS) elements in the PH05 promoter (UASpl and UASp2, see Fig. 1)7, which are critically required for J. Svarenis at the Departmentof Pathology, Washington UniversitySchoolof Medicine, St Louis, MO 63110-1093, USA;and W. H6rz is at the Institut ffir PIlysiologische Chemie, Universit/~tMiinchen,Schillerstr. 44, D-80336 Minchen, Germany. Email: [email protected] .de
promoter activation8, Pho2 binds to several sites in the PH05 promoter, one of_~-Alichoverlaps with the Pho4-hinding site at UASpl, whte another two sites flank the second Pho4-binding site in UASp2 (ReL 9). Pho2 binds cooperatively with Pho4 at both Pho4-binding sites, probably as a result of a direct interaction between Pho2 and a specific domain of Pho4 (Ref. 10). Pho2 plays a similar role in the HO promoter, where it binds cooperatively with Swi5 (Ref. I l). Two negative regulators of the PHO system, Pho80 and Pho85, form a kinase complex, related to cyclin-CDK (cyclindependent kinase) complexes, which can phosphorylate Pho4 in repressive (high phosphate) conditions12.The regulation of the Pho80-Pho85 complex and the intracellular signalling of the PHO system have been excellently reviewed in a recent article ~3.
Nucleosomestructure and PH05 regulation The repressed PH05 gene is packaged in a positioned array of nucleosomes that is interrupted only in the promoter region by a short stretch of 80 base pairs (hp)14. Upon induction of PH05 transcription by phosphate starvation, a 600 bp region of the PH05 promoter becomes hypersensitive to nucleases, reflecting a profound alteration of the structure of four nucleosomes|5 (Fig. 2). Although this region is no longer protected from nuclease digestion, it is possible that histones remain associated with the DNA in a configuration that increases its accessibility not only to various nucleases, but also to binding of Pho4 and other transcription factors. For example, loss of the histone H2A-H2B dimers from the nucleosome has been
shown to increase acceJsiblity of tLe DNA that wraps around the remaining H3-H4 tetramer ~6. The nucleosomat configuration that overRays the PH05 regulatory elements and its disruption upon promoter activation provokes several questions. Me nucleosomes required for repression of tile promoter under high phosphate conditions? Must the nuc!eosomes be removed or altered to allow PH05 tram scription? How does nucleosome structure affect binding of Pho4, Pho2 and the transcription initiation complex? Is nucleosome disruption a consequence or rather a necessary prerequisite for transcription initiation? How is the nucleosome structure of the repressed PH05 promoter set up? In an effort to shed light on the role of nucleosomes in PH05 regulation, we have replaced the DNA sequence corresponding to nucleosome -2 with two foreign DNAsegments of the same length: a fragment from the Mrican green monkey c~-satelliteDNA,which is Imown to assocF ate with histones to give a uniquely positioned nucleosome or, alternatively, a fragment derived from pBR322 DNA~L The satellite fragment forms a nucleosome that persists under inducing conditions. At the same time, the inducibility of this PH05 promoter variant is severely reduced compared to the wildtype version. By contrast, the pBR322 fragment makes the promoter weakly constitutive, and induction proceeds to levels even higher than with a promoter lacking an insert. Analysis of the chromatin structure at non-inducingconditions reveals a nucleosome on the pBR seg~ ment that is removed upon induction. These results argue that the quality of the histone-DNA interactions at the promoter is a determinant of the bas:,J and activated levels ol PH05 activity A complementary approach in which the histone, rather than the DNA component of the nucleosome, was altered, was taken by Grunstein and colleagues. They created yeast strains in which nucleosome formation was effectively prevented by shutting off synthesis of one of the histones. These experiments show that the resulting depletion of nucleosomes in the genome leads to activation of the PH05 promoter TM. Subsequent experiments showed that this effect requires the presence of only the TATAelements of the promoter, but not the UAS elements 19. Therefore, it was suggested that the presence of nucleosome -1 inhibits formation of the transcription fnRiation complex on the
Copyright~ i997,ElsevierScienceLtd.Allrightsr~erved.0968-0004/97/$1700 PiI:S0968.Dfl04(97)91001-3
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-4
-3
-2
-t
+ P~
- P~
@ UASpl UASp2
Rgum ~. Chromatin structure at the PH05 promoterunder nonqnduced(+ P3 and induced (-P3 conditions. Nucleosomes -1, -2, -3 and -4 are removed upon activation. The smail circles mark UASpl (open) and UASp2 (solid), which are Pho4-bindingsites found by in vitro and in vivo footprinting experiments. The positions are listed relative to the coding sequence (thick blue line). PHO$ TATA box. Activation of the pro-
moter under conditions of histone depletion does not reach the level obtained during physiological activation, however, indicating that PH05 activation is not solely the result of alleviating nueleosomal repression at the TATA box. llfwqll~ff of transcdption factor binding and nucleosome structure We have recently complemented our analyses of the chromatin structure o! the PH05 promoter, by in vivo footprintlng experiments, to determine directly if a Pho4-blnding site Is occupied under various conditions. Upon PH05 activation and concomitant nucleosome disruption, there Is Pho4 binding to both UAS elements, but no binding is observed at high phosphate (repres~sing)conditions2°. These experiments directly show that the binding capacity of Pho4 Is strongly increased after phosphate starvat~or~. The lack of Pho4 binding to the repressed PH05 promoter is consistent with the observation that Pho4 is localized predominantly In the cyt,,plasm under high phosphate conditions m.
As the in vivo binding capacity of Pho4 is much greater after activation of the PHO system 0.e. low phosphate or in a pho80 strain), we sought to ascertain how Pho4 can interact with a nucleosomal site under these conditions. Such an experiment is complicated by the fact that low phosphate conditions normally result in pucleosomal disruption of the wild-type PHO5 promoter. However, mutation of UASpl results in a prorooter in which the nucleosome structure persists even after phosphate starvation. In this mutant promoter, there is no binding of 'activated' Pho4 to UASp2 as schematically shown in Fig. 3b (Ref. 20).
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Therefore, nucleosome -2 hinders binding of Pho4 even under activating conditions. However, nucleosome-2 is apparently not an absolute barrier to Pho4 binding, in the promoter in which UASpl is mutated, overproduction of Pho4 will restore its binding to UASp2 (Fig. 3c) and will also disrupt the same four nucleosomes that are affected by activation of the wild-type promoter'-'°.Studies using other variants of the PH05 promoter and of the Pho4 protein itself invariably show that, when chromatin opening occurs, all four nucleosomes are disrupted in an 'a!~ or nothing' fashion. These four nucleosomes might form a chromattn microdomaio that is coordinately disrupted by the action of Pho4. The clearest evidence for the nudeosomal inhibition of Pho4 binding to UASp2 stems from experiments that took advantage of Pho4 variants that can bind DNA, but cannot disrupt nucieosome stru-ture. Such Pho4 variants can bind UAbpl within the hypersensitive site, but do not bind UASp2 within nucleosome -2 (Fig. 3e)21. In order to eliminate the possibility that the Pho4 variants bind to UASpl with greater affinity than to UASp2, we used a version of the PHO5 promoter In which the positions of the two UAS elements were switched. This experiment revealed that the Pho4 variants could bind UASp2 if it was situated within the nucleosome-free region (Fig. 30. Although there is clear evidence, mostly from in vitro experiments, that certain factors such as Gal4 and the glucocorticoid receptor can bind to a nueleosome to form a ternary complex (factor-histones-DNA)22. 23, we have not obtained any evidence that Pho4 can bind stably to a nucleosomal site in vivo.
Surprisingly, moving UASp2 to the exposed hypersensitive site in the reversed PH05 promoter was found to allow binding of a related bHLH protein, Cpfi (Ref. 20), which had previously been shown to have an overlapping binding specificity witla Pho4. A s~milar situation exists in the native PH08 promoter in which the UASp2 element lies in a constitutively nucleosome4ree region24 and is at least partially bound by Cpfl at high phosphate. Therefore, one consequence of the position of UASp2 within nucleosome -2 is that binding of a related protein, Cpfl, is prevented, indicating that nucieosome structure can play a role ~n the binding selectivity of UAS elements by ~estricting b~nding to a single member of a ~amily ~f related DNA-binding proteins. M0dei for P•05 activati~¢~ Although nucleosomes inhibit factor binding, activation ot Pho4 by phosphate starvation initiates a process that overcomes nucleosomal repression, thereby facilitating formation of transcription initiation complexes. The two most obvious mechanisms that would explain chromatin disruption at the PH05 promoter would be that it is the conse-. quence of processes occurring during replication or transcription itself. In the first model, activation of PH05 transcription requires passage through S phase In order that Pho4 can establish binding on the Hg)5 promoter during the nucleosome remodelling that accompanies DNA replication. However, expeflments using a temperature~sensitive allele of HI080 show that DNA replication is not required [or nudeosome disruption to occur 25. The second model is that chromatin disruption is caused by the increased initiation of transcription of the PH05 gene. Some experiments indicate that RNA polymerase leaves a trail of negative supercoiling in its wake, which could potentially alter nucleosome structure. However, mutation o| the TATAbox in the PH05 promoter reduces transcription to almost undetectable levels, but does not prevent the chromatin transition, thereby showing that active trans~:ription is not the cause of nucleosome disruptionz~. Becanse of the strict requirement of Pho4 for activation of the PH05 promoter, it is reasonable to conclude that the first step in chromatin disruption is Pho4 binding. At normal levels of Pho4, both Pho4 binding sites are required for PH05 promoter activation. Mutation of either UASpl or UASp2 prevents the
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chromatin transition and reduces promoter strength to tess than 10% of the wild-type promote~ ~. The presence o~ several Pho4-binding sites suggests that they cooperatively activate the PH05 promoter. However, activation by Pho4 is not cooperative when two b~nding sites are placed upstream ~ the CYCt promoter, but rather is proportional to the number of binding sites a~. Although the two UAS elements do not appear to activate transcription coope~'atively, it is possible that they ~nteract to disrupt ~ue~eosome structure.
@ 0-~ ( ~ - 2 >-S
Clal BamHl
As described above, nucleosome -2 interferes with Pho4 binding to UASp2, and therefore it is likely that Pho4 (or Pho4 together with Pho2) binds initially to UASpl w~thin the hypersensitive site. Figure 2 Experiments using truncated versions of Analysis of the chromatin transition at the PH05 promoter by DNasel. Nuclei from cellt Pho4 reveal that the next steps in nucleogrown under high phosphate (left) or no phosphate conditions (right) were digested with in some disruption require the activation creasing concentrations of DNaseL [)NA was isolated, digested with Apal, and DNase~ domain of Pho4 (Ref. 21). Similar results cleavage analysed by the indirect endqabeling protocol21,22. The perditions of the BamH in the GALl promoter show that the and Clal restriction sites in the promoter are shown in the center lanes by doubte digests activation domain of Gal4 is required for of genomic DNA with Apal/Clal end Apat/BamHI. The short hypersensitive site containing UASpl, which is characteristic of the repressed state (see Fig. 1l, is marked by an arrow. disruption of a nucleosome adiacent to a The schematic on the left shows the positioned nucleosomes present at high phosphate cluster of Gal4~bindings~tesa~ Moreover, conditions and is drawn to scale with the gel, Nucleosames -1 to -4, which are disrupted nucleosome disruption is not a unique upon PH05 activation, are pale yellow circles as in Fig. 1. property of the Pho4-activation domain, because a Pho4 derivative containing the VPl6-activation domain in place of is an integral part of its activation do- were recently misinterpreted in a rett=e native domain is also competent to main. As activation domains have not view where it was stated that nucleodisrupt PH05 chromatin structure ~. been shown to directly alter nucleosome some disruption at the P t f 0 5 promoter structure in purified in vitro systems, it occurs as the consequence of transcripis likely that nucleosome disruption is tional activation~. Actually, the opposite P~echanis~n of aac~eosomedisruption The experiments described above in- eifected by interactions with other cel- is true, chromatin disruption [s observed dicate that activation domains are re- lular proteins and/or complexes. Acidic in the complete absence of transcripquired not only to interact with com- activation domains have been shown to tion, and all results so far are consistent ponents of the basal transcriptional interact with several components oi the with the conclusion that the chromatin apparatus, hut also to mediate ciwomatin basal transcriptional machinery. More disruption at the promoter is the prechanges in the promoter, which facilitates recently, it has been shown that RNA requisite for transcription of the PH05 transcription i~itiation. This raises the polymerase li and many of the basal gone rather than its consequence. Although we have demonstrated that question of whether these two processes transcription [actors, including a group can be functionally separated. In other of Srb proteins and Gall 1, exist in a large recruitment of the holoenzyme to the PIt05 promoter can trigger nucleosome systems, there are several examples of complex termed the holoenzyme3°,3t. Experiments by Barberis et al. 32 show disruption, it is not clear whether nucleopruteins that can open chromatin structure, but do not themselves activate that fusion of a holoenzyme component, some disruption is intrinsic to hoiotranscription to a significant degree. Galll, to a DNA-binding domain pro- enzyme components that have so far Binding sites for such proteins generally duces a particularly powerful activator been identified. The SWI-SNFcomplex, activate transcription little when they by directly recruiting the holoenzyme which has recently been proposed to b~ are placed alone in a promoter, but to the target promoter in the absence o[ associated with the holoenzyme3s, is a greatly accentuate the activation of ad- a classical activation domain. We de- prime candidate for contributing such jacent enhancing sequences, presumably cided, therefore, to examine if recruiting an activity. Several in vitro studies using by providing a chromatin environment the holoenzyme to the PH05 promoter both yeast and human versions o[ this in which other activators can [unction through a Pho4 DNA-binding domain complex have shown that this complex could disrupt nucleosomes. To do so, can catalyse an ATP-dependent disordermore efficiently zo. [n the case of PH05, we can obtain we replaced the Pho4-activation domain ing of nucleosome structure that facili.. chromatin opening in the absence of with different components o! the RNA tares binding of transactivators anc~ transcription by mutating the TATAbox, polymerase II holoenzyme. Hybrid pro- TATA-binding protein3~8. Interestingly, as described above. However, we have teins hearing the DNA-bindingregion of nucleosome disrupticn at the PH05 pronot found a Pho4 derivative or mutant Pho4 fused to Gall 1 or Srb2 can remodel moter On a phoSO~' strain) requires that can open chromatin without acti- chromatin at the PH05 promoter, even glucose, but no other component of the vating transcription, suggesting that the with a TATA-deletedpromoter that can- growth medium, while nucleosome rechromatin reorganization activity of Pho4 not support transcription33. Our results formation proceeds in the absence of
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of a PH05 gene copy integrated into the URA3 locus, while the endogenous Pt105 Pho. gene remained repressed 4~. This indicates that the SIN4 gene product could be involved in insulating a promoter from its chromosomal environment. Consistent with this interpretation, we find that certain foreign DNA sequences in~UASpl ~ Pho4 Aact serted in the vicinity of the endogenous PH05 gene alleviate PH05 repression at high phosphate in a sin4 (but not in a wild-type) background, concomitant with a loss of nucleosome positioning over the promoter. Histone acetylaUon has been proAUASpl Pho4 Aact posed by many in the chromatin field to play a role in making nucleosomes more accessible to factor binding. This past year has marked a milestone in the chromatin field in that the elusive proteins that are responsible for histone acetylation and deacetylation have begun to be identified, as well as the protein Pho4 binding to UAS elements at the PH05 promoter. On the left, binding of Pho4 in vivo motifs that catalyse these processe~. (a) to the wild-type PH05 promoter and (b, ¢} to a variant promoter lacking UASpl is Satisfyingly, Gcn5, the protein that shown. (¢) is under conditions of Pno4 overproduction. On the right, in vivo binding of catalyses histone acetytatio# ?, was prePho4 (d) with or (e, f) without the activation domain, depicted as an oval, to the two UAS viously identified as a component of a elements at the PH05 promoter in their (d. e) natural and (f) reverse orientation is shown. coactivator complex required for the full The green arrow denotes initiation of transcription. activation potential of acidic activation domains4s. Conversely, a protein that glucose, suggesting that the disruption to attain true phosphate starvation con- had been identified as playing a role in of the nucleosomes is ATP-dependent 2~. ditions. Any mutation impairing growth silencing, Rpd3, has now been identiit was originally reported that a snt2 can therefore interfere with PH05 de- fied as a histone deacetylase49. Several mutation prevented activation of the repression without being directly in- years ago, it was shown that in an rpd3 PH05 gene39. We have found, however, volved in the activation of the PHO5 pro- mutation, neither lull repression nor that a deletion of the SNF2 gene, which motet. For tMs reason, we pregrow ceils full derepression of the PH05 gene is in other experiments has been shown under high phosphate conditions and achieved, leading to a diminished amplito disable SWI-,SNFfunction, has only a then shift them into a synthetic medium tude of regulation'% Defining the preminor effect on PHO5 activation and lacking phosphate altogether, rather cise role of such chromatln modifiers in chromatin disruption at the PH05 pro- than using low phosphate medium. PH05 regulation will further refine our moter~. However, there are several picture of how chromatin structure condistinct activities capable of labilizing Novel m ~ l a t m s of the PH05 trois gene activation. histone-DNAinteractions, and they might Evidence that the SWJ-SNF complex prove to be components of the holo- functions through chromatin structure Conclusions enzyme or at least transiently associate came originally from the isolation of It has become abundantly clear that with it. An example of such an activity bypass suppressor mutations called sin nucleosome structure is not merely an from Drosophila (NURF, nucleosome- (switch independent). The sinl suppres- ancestral packing material, but instead remodeling factor) 4°does not contain the sor mutant lies in a gene that bears is a dynamic structure that is exploited Drosophila homologue of Snf2, brahma, some similarity to HMG-I (Ref. 42), an by the cell for complex differential regubut instead contains another member of abundant chromosomal protein. The sin2 lation of gene activity. A corollary of this the Snf2 family, called iswi41. Given the suppressor is a mutant in one of the idea is that the nucleosome is not a number of Snf2-related proteins in yeast two copies of the histone H3 gene 43. constant, but rather can play a number identified by the yeast genome project, One interesting member of the Sin family of roles in gene regulation depending there could be a family of such activities of regulators is the sin4 suppressor mu- on its structure, modification state and that might play at least partially redun- tation~. The phenotype of this mutant chromosomal context. Our work on dant roles. is similar to yeast in which histone syn- PH05 indicates that chromatin has a reThe seemingly contradictory data on thesis is inhibited (see above). First, sev- pressive effect on th ,~promoter and that the role of Sn[2 in PH05 regulation eral promoters lacking UAS elements are chromatin opening is an integral part of might have to do with the fact that the activated in this mutant strai# 5. Second, the transcriptional activation process. A low phosphate medium, which is often the superhelical densities of yeast play regulatory role for chromatin is corrobouse(i to induce the PHO system, con- mids are decreased, which could indicate rated by a wealth of genetic evidence tains phosphate in concentrations high inhibition of nucleosome assembly44. It showing that the growing list of factors enough to repress PH05, and phosphate was recently reported that a sin4 mutation that regulate transcription includes chromust be consumed by growth of a culture leads to Pho4-independent derepression matin components (histories and HMG wi PHO5 pmmotor ~
(a) ~ - ~ - -
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proteins) as well as proteins that modify chromatin (acetylases and deacetylases). The recent completion 9f the yeast genome sequence has aided ia the idefltification of p r o t e i n s that c o u l d potentially modify chromatin structure, and efforts in this field will become inc r e a s i n g l y ~ocused on how these proteins determine promoter activity.
Aok~ow~e~e[ae~ts This work was supported by the Deutsche Forschungsgeme~nschaft (Sl~ 199) and Fends der Chemischen lndustrie. RefereJlces I Vogel, K.and Hinnen, A. (1990) Mol. Microbial. 4, 2013-2017 2 Ve~ter, U. a~d Horz. W. (1989) Nucleic Acids Res. 17, 1353-1369 3 Koren. R., LeVitre, J. and Bostian, K. A. (1986) Gene 41,271-280 4 8erben, G., Legrain, M., Gilliquet, V. and Hilger, F. (1990) Yeast 6,451--454 5 Sengstag, C. and Hinnen, A. (1987) Nucleic Acids Res. 15, 233-246 6 Berben, G., Legrain, M. and Hilger, F. (1988) Gene 66, 307-312 7 Vogel K., Hdrz, W. and Hinnen, A. (1989) MoL Celt, Biol. 9, 2050-2057 8 Rudolph, H. and Hinnen, A. (1987) Prec. Natl. Acad. Sci. U. S. A. 84, 1340-1344 9 Barbaric, S.. MSnsterkdtte~, M,, Svaren, J. and H~rz, W. (1996) Nucleic Acids Res. 24,
4479-4486 10 Hirst, K., Fisher, F., McAndrew, P. C. and Goding, C. R. (19941 EMBO 3. 13, 5450-5420 I 1 6razas, R. M and Stillmat~, D. J. (1993) Mol. Celt. Biol. 3.3, 5524-5537, correction 7200 12 Kaffman, A.. Herskowitz, I., Tjian, R. and O'Shea, E. K. (1994) Scie~Jce 263, 1153-1156 13 Lenburg, M. E. and O'Shea, E. K. (1996) Trends Biocl~em. Sci. 21, 383-387 14 Almer, A. and Hd/z, W. (1986) EMBO J. 5,
2881- 2687 15 Almer, A., Rudolph, H., Hinnen, A. and H6rz, W. (!986} EMBO J. 5, 2689~--2696 16 Hayes, J. J. and Wolffe, A, P. (1992) Prec. Natl. Acad. Sci. U. S. A. 89, 1229-1233 17 Straka, C. and H6rz, W. (1991) EMBOJ. 10. 361-368 18 Han, M., Kim, U. £, Kayne, P. and Grunstein, M. (1988} EMBO ./. 7, 2221-2228 19 Han, M. and Grunstein, M. (1988) Cell 55, 1137-1145 20 Venter, U. et at. (1994) EMBO J. 13, 4848-4855 21 Svaren, J., Schmitz, J. and H6rz, W. (1994) EMBO J. 13, 4856-4862 22 Pedmann, T. and Wrange, O. (1988) EMBO J. 7, 3073-3079 23 Adams, C. C. and Workman, J. L. (1993) Cell 72, 305-308 24 Barbaric, S., Fascher, K. D. and H6rz, W. (1992) Nucleic Acids Res. 20, 1031-1038 25 Schmid, A., Fascher, K. D. and H6rz, W. (1992) Celt 71, 853-864 26 Fascher, K. D., Schmitz, J. and H6rz, W. (1993) J. MoL Biol. 231, 658-667 27 Sengstag. C. and Hinnen, A. (1988) Gene 67, 223--228 28 Axelrod, J. D., Reagan, M. S. and Majors, J. (1993) Genes Dev. 7,857-869
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derivatives) that were shorter than the sum of the van der Waals radii of the atoms involved. Furthermore, in 1982, a survey of numerous neutron structures conclusively established the existence 9f C-H,,.Y hydrogen bonds4. The strongest hydrogen bond is the C-H.,oO contact, which exhibits typical directional and electrostatic features (Fig. 1). C-H°°.O bond strengths often correlate with the Observations of short C-H...O contacts in biological macromolecules, includacidity of the hydrogen attached to the ing nucleic acids, proteins and carbohydrates, suggest that ttlese unconvencarbon atom, with methyne and methyltional hydrogen bonds have both a structurally and functionally important role. ene groups being stronger donors than methyl groupss. Carbons with adjacent HYDROGEN BONDS, ARE one of the aqueous environment and ligands. They nitrogen atoms form particularly strong most important types of interactions in are attractive electrostatic interactions hydrogen bonds4. High-precision neutron molecular biology, shaping not only of the type X-H°°oY, in which Y, the diffraction structures and theoretical the three-dimensional structures of hydrogen-bond acceptor, carries a full studies indicate that the covalent C-H macromolecules, but also influencing or partial negative charge, while the bond lengthens when the hydrogen atom their interactions with the surrounding hydrogen-bond donor, X, is more negative is involved in a hydrogen bond contact~ . The surface of macromolecules conthan H. To maximize the electrostatic attraction, the hydrogen preferentially sists mainly of groups that can act either M, C, Wahl is at the Max-Planck-lnstitut for approaches Y along the direction of and as donors or as acceptors of C-H°o.O/N Biochemie, Abteilung Strukturforschung, in a plane with a lone-pair orbital of Y hydrogen bonds. Until recently, many Am Klopferspitz 18a, 1:)432512 PlaneggMartinsried, Germany; and M. Sundarallngam (see Ref. 1 for a review). Sutor first sum- cases of C-Ho..O interactions were classiis at the Laboratory of Biological marized the crystallographic evidence fied as hydrophobic interactions and Macromolecular Structure, Departments of in support of the existence of C-H...O not properly identified. Owing to the Chemistry and Biochemistry, The Ohio State hydrogen bonds2,3. She observed that l ~ e size of the macromolecules and the University, 120 West 18th Avenue Columbus, there were contacts in some crystal limited resolution, X-ray studies cannot OH 43210-1002, USA. structures (of nucleic acid bases and locate hydrogen atoms. While C-H.o°O Email: [email protected]
C=H.,oOhy&'ogenbondingin biology Markus C. Wahl and MuttaiyaSundaralingam
Copyright© 1997,ElsevierScienceLtd.Allrightsreserved.0968-0004/97/$17.00 PII:S0968-0004(97)01004-9
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Tran c plon adors vs aac eosomes: reg laloa of the PHO$ # omote yeast John Svaren arid Wolfram HSrz Activation of the Saccharomyces cerevisiae PH05 gene is accompanied by the disruption of four positioned nucleosomes at the promoter. The chmmatin transition requires a DNA-binding protein, Pho4, and its tmnsactivation domain. The mechanism of nucleosome disruption and the contribution of the nucleosemes to PH05 regulation are reviewed.
ONE OF THE main challenges in the area of transcriptional regulation is to discover how signalling pathways impinge on the activity of DNA-binding proteins to alter patterns of gene expression. A success story in this regard has been the PHO system, which is involved in regulating phosphate metabolism in the cell and is poised to scavenge extracellular phosphate when internal pools become depleted. Based on the pioneering genetic studies of Oslima and coleagues, the PHO system has provided fettle ground for investigations of sigual transduction, transcriptional regulation and the function of chromatin structure. M0du[at0rs of PHO tmnscipt[0n Phosphate starvation o| the yeast Saccharomyces cerevisiae results in at least a 50-fold increased production of secreted acid phosphatase, which is composed of three isozymes produced from the PH05, PHOIO and PHOII genes t2. More than 90% of the acid phosphatase activity is supplied by the PH05 gene product, and experiments have therefore concentrated on its regulation. Positive modulators of PH05 transcription include Pho4 (Ref. 3), a basic helix-loophelix (bHLH) transactivator4, and Pho2 (Ref. 5), a homeobox DNA-binding protein6. Pho4 binds to two upstream activating sequence (UAS) elements in the PH05 promoter (UASpl and UASp2, see Fig. 1)7, which are critically required for J. Svarenis at the Departmentof Pathology, Washington UniversitySchoolof Medicine, St Louis, MO 63110-1093, USA;and W. H6rz is at the Institut ffir PIlysiologische Chemie, Universit/~tMiinchen,Schillerstr. 44, D-80336 Minchen, Germany. Email: [email protected] .de
promoter activation8, Pho2 binds to several sites in the PH05 promoter, one of_~-Alichoverlaps with the Pho4-hinding site at UASpl, whte another two sites flank the second Pho4-binding site in UASp2 (ReL 9). Pho2 binds cooperatively with Pho4 at both Pho4-binding sites, probably as a result of a direct interaction between Pho2 and a specific domain of Pho4 (Ref. 10). Pho2 plays a similar role in the HO promoter, where it binds cooperatively with Swi5 (Ref. I l). Two negative regulators of the PHO system, Pho80 and Pho85, form a kinase complex, related to cyclin-CDK (cyclindependent kinase) complexes, which can phosphorylate Pho4 in repressive (high phosphate) conditions12.The regulation of the Pho80-Pho85 complex and the intracellular signalling of the PHO system have been excellently reviewed in a recent article ~3.
Nucleosomestructure and PH05 regulation The repressed PH05 gene is packaged in a positioned array of nucleosomes that is interrupted only in the promoter region by a short stretch of 80 base pairs (hp)14. Upon induction of PH05 transcription by phosphate starvation, a 600 bp region of the PH05 promoter becomes hypersensitive to nucleases, reflecting a profound alteration of the structure of four nucleosomes|5 (Fig. 2). Although this region is no longer protected from nuclease digestion, it is possible that histones remain associated with the DNA in a configuration that increases its accessibility not only to various nucleases, but also to binding of Pho4 and other transcription factors. For example, loss of the histone H2A-H2B dimers from the nucleosome has been
shown to increase acceJsiblity of tLe DNA that wraps around the remaining H3-H4 tetramer ~6. The nucleosomat configuration that overRays the PH05 regulatory elements and its disruption upon promoter activation provokes several questions. Me nucleosomes required for repression of tile promoter under high phosphate conditions? Must the nuc!eosomes be removed or altered to allow PH05 tram scription? How does nucleosome structure affect binding of Pho4, Pho2 and the transcription initiation complex? Is nucleosome disruption a consequence or rather a necessary prerequisite for transcription initiation? How is the nucleosome structure of the repressed PH05 promoter set up? In an effort to shed light on the role of nucleosomes in PH05 regulation, we have replaced the DNA sequence corresponding to nucleosome -2 with two foreign DNAsegments of the same length: a fragment from the Mrican green monkey c~-satelliteDNA,which is Imown to assocF ate with histones to give a uniquely positioned nucleosome or, alternatively, a fragment derived from pBR322 DNA~L The satellite fragment forms a nucleosome that persists under inducing conditions. At the same time, the inducibility of this PH05 promoter variant is severely reduced compared to the wildtype version. By contrast, the pBR322 fragment makes the promoter weakly constitutive, and induction proceeds to levels even higher than with a promoter lacking an insert. Analysis of the chromatin structure at non-inducingconditions reveals a nucleosome on the pBR seg~ ment that is removed upon induction. These results argue that the quality of the histone-DNA interactions at the promoter is a determinant of the bas:,J and activated levels ol PH05 activity A complementary approach in which the histone, rather than the DNA component of the nucleosome, was altered, was taken by Grunstein and colleagues. They created yeast strains in which nucleosome formation was effectively prevented by shutting off synthesis of one of the histones. These experiments show that the resulting depletion of nucleosomes in the genome leads to activation of the PH05 promoter TM. Subsequent experiments showed that this effect requires the presence of only the TATAelements of the promoter, but not the UAS elements 19. Therefore, it was suggested that the presence of nucleosome -1 inhibits formation of the transcription fnRiation complex on the
Copyright~ i997,ElsevierScienceLtd.Allrightsr~erved.0968-0004/97/$1700 PiI:S0968.Dfl04(97)91001-3
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-4
-3
-2
-t
+ P~
- P~
@ UASpl UASp2
Rgum ~. Chromatin structure at the PH05 promoterunder nonqnduced(+ P3 and induced (-P3 conditions. Nucleosomes -1, -2, -3 and -4 are removed upon activation. The smail circles mark UASpl (open) and UASp2 (solid), which are Pho4-bindingsites found by in vitro and in vivo footprinting experiments. The positions are listed relative to the coding sequence (thick blue line). PHO$ TATA box. Activation of the pro-
moter under conditions of histone depletion does not reach the level obtained during physiological activation, however, indicating that PH05 activation is not solely the result of alleviating nueleosomal repression at the TATA box. llfwqll~ff of transcdption factor binding and nucleosome structure We have recently complemented our analyses of the chromatin structure o! the PH05 promoter, by in vivo footprintlng experiments, to determine directly if a Pho4-blnding site Is occupied under various conditions. Upon PH05 activation and concomitant nucleosome disruption, there Is Pho4 binding to both UAS elements, but no binding is observed at high phosphate (repres~sing)conditions2°. These experiments directly show that the binding capacity of Pho4 Is strongly increased after phosphate starvat~or~. The lack of Pho4 binding to the repressed PH05 promoter is consistent with the observation that Pho4 is localized predominantly In the cyt,,plasm under high phosphate conditions m.
As the in vivo binding capacity of Pho4 is much greater after activation of the PHO system 0.e. low phosphate or in a pho80 strain), we sought to ascertain how Pho4 can interact with a nucleosomal site under these conditions. Such an experiment is complicated by the fact that low phosphate conditions normally result in pucleosomal disruption of the wild-type PHO5 promoter. However, mutation of UASpl results in a prorooter in which the nucleosome structure persists even after phosphate starvation. In this mutant promoter, there is no binding of 'activated' Pho4 to UASp2 as schematically shown in Fig. 3b (Ref. 20).
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Therefore, nucleosome -2 hinders binding of Pho4 even under activating conditions. However, nucleosome-2 is apparently not an absolute barrier to Pho4 binding, in the promoter in which UASpl is mutated, overproduction of Pho4 will restore its binding to UASp2 (Fig. 3c) and will also disrupt the same four nucleosomes that are affected by activation of the wild-type promoter'-'°.Studies using other variants of the PH05 promoter and of the Pho4 protein itself invariably show that, when chromatin opening occurs, all four nucleosomes are disrupted in an 'a!~ or nothing' fashion. These four nucleosomes might form a chromattn microdomaio that is coordinately disrupted by the action of Pho4. The clearest evidence for the nudeosomal inhibition of Pho4 binding to UASp2 stems from experiments that took advantage of Pho4 variants that can bind DNA, but cannot disrupt nucieosome stru-ture. Such Pho4 variants can bind UAbpl within the hypersensitive site, but do not bind UASp2 within nucleosome -2 (Fig. 3e)21. In order to eliminate the possibility that the Pho4 variants bind to UASpl with greater affinity than to UASp2, we used a version of the PHO5 promoter In which the positions of the two UAS elements were switched. This experiment revealed that the Pho4 variants could bind UASp2 if it was situated within the nucleosome-free region (Fig. 30. Although there is clear evidence, mostly from in vitro experiments, that certain factors such as Gal4 and the glucocorticoid receptor can bind to a nueleosome to form a ternary complex (factor-histones-DNA)22. 23, we have not obtained any evidence that Pho4 can bind stably to a nucleosomal site in vivo.
Surprisingly, moving UASp2 to the exposed hypersensitive site in the reversed PH05 promoter was found to allow binding of a related bHLH protein, Cpfi (Ref. 20), which had previously been shown to have an overlapping binding specificity witla Pho4. A s~milar situation exists in the native PH08 promoter in which the UASp2 element lies in a constitutively nucleosome4ree region24 and is at least partially bound by Cpfl at high phosphate. Therefore, one consequence of the position of UASp2 within nucleosome -2 is that binding of a related protein, Cpfl, is prevented, indicating that nucieosome structure can play a role ~n the binding selectivity of UAS elements by ~estricting b~nding to a single member of a ~amily ~f related DNA-binding proteins. M0dei for P•05 activati~¢~ Although nucleosomes inhibit factor binding, activation ot Pho4 by phosphate starvation initiates a process that overcomes nucleosomal repression, thereby facilitating formation of transcription initiation complexes. The two most obvious mechanisms that would explain chromatin disruption at the PH05 promoter would be that it is the conse-. quence of processes occurring during replication or transcription itself. In the first model, activation of PH05 transcription requires passage through S phase In order that Pho4 can establish binding on the Hg)5 promoter during the nucleosome remodelling that accompanies DNA replication. However, expeflments using a temperature~sensitive allele of HI080 show that DNA replication is not required [or nudeosome disruption to occur 25. The second model is that chromatin disruption is caused by the increased initiation of transcription of the PH05 gene. Some experiments indicate that RNA polymerase leaves a trail of negative supercoiling in its wake, which could potentially alter nucleosome structure. However, mutation o| the TATAbox in the PH05 promoter reduces transcription to almost undetectable levels, but does not prevent the chromatin transition, thereby showing that active trans~:ription is not the cause of nucleosome disruptionz~. Becanse of the strict requirement of Pho4 for activation of the PH05 promoter, it is reasonable to conclude that the first step in chromatin disruption is Pho4 binding. At normal levels of Pho4, both Pho4 binding sites are required for PH05 promoter activation. Mutation of either UASpl or UASp2 prevents the
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chromatin transition and reduces promoter strength to tess than 10% of the wild-type promote~ ~. The presence o~ several Pho4-binding sites suggests that they cooperatively activate the PH05 promoter. However, activation by Pho4 is not cooperative when two b~nding sites are placed upstream ~ the CYCt promoter, but rather is proportional to the number of binding sites a~. Although the two UAS elements do not appear to activate transcription coope~'atively, it is possible that they ~nteract to disrupt ~ue~eosome structure.
@ 0-~ ( ~ - 2 >-S
Clal BamHl
As described above, nucleosome -2 interferes with Pho4 binding to UASp2, and therefore it is likely that Pho4 (or Pho4 together with Pho2) binds initially to UASpl w~thin the hypersensitive site. Figure 2 Experiments using truncated versions of Analysis of the chromatin transition at the PH05 promoter by DNasel. Nuclei from cellt Pho4 reveal that the next steps in nucleogrown under high phosphate (left) or no phosphate conditions (right) were digested with in some disruption require the activation creasing concentrations of DNaseL [)NA was isolated, digested with Apal, and DNase~ domain of Pho4 (Ref. 21). Similar results cleavage analysed by the indirect endqabeling protocol21,22. The perditions of the BamH in the GALl promoter show that the and Clal restriction sites in the promoter are shown in the center lanes by doubte digests activation domain of Gal4 is required for of genomic DNA with Apal/Clal end Apat/BamHI. The short hypersensitive site containing UASpl, which is characteristic of the repressed state (see Fig. 1l, is marked by an arrow. disruption of a nucleosome adiacent to a The schematic on the left shows the positioned nucleosomes present at high phosphate cluster of Gal4~bindings~tesa~ Moreover, conditions and is drawn to scale with the gel, Nucleosames -1 to -4, which are disrupted nucleosome disruption is not a unique upon PH05 activation, are pale yellow circles as in Fig. 1. property of the Pho4-activation domain, because a Pho4 derivative containing the VPl6-activation domain in place of is an integral part of its activation do- were recently misinterpreted in a rett=e native domain is also competent to main. As activation domains have not view where it was stated that nucleodisrupt PH05 chromatin structure ~. been shown to directly alter nucleosome some disruption at the P t f 0 5 promoter structure in purified in vitro systems, it occurs as the consequence of transcripis likely that nucleosome disruption is tional activation~. Actually, the opposite P~echanis~n of aac~eosomedisruption The experiments described above in- eifected by interactions with other cel- is true, chromatin disruption [s observed dicate that activation domains are re- lular proteins and/or complexes. Acidic in the complete absence of transcripquired not only to interact with com- activation domains have been shown to tion, and all results so far are consistent ponents of the basal transcriptional interact with several components oi the with the conclusion that the chromatin apparatus, hut also to mediate ciwomatin basal transcriptional machinery. More disruption at the promoter is the prechanges in the promoter, which facilitates recently, it has been shown that RNA requisite for transcription of the PH05 transcription i~itiation. This raises the polymerase li and many of the basal gone rather than its consequence. Although we have demonstrated that question of whether these two processes transcription [actors, including a group can be functionally separated. In other of Srb proteins and Gall 1, exist in a large recruitment of the holoenzyme to the PIt05 promoter can trigger nucleosome systems, there are several examples of complex termed the holoenzyme3°,3t. Experiments by Barberis et al. 32 show disruption, it is not clear whether nucleopruteins that can open chromatin structure, but do not themselves activate that fusion of a holoenzyme component, some disruption is intrinsic to hoiotranscription to a significant degree. Galll, to a DNA-binding domain pro- enzyme components that have so far Binding sites for such proteins generally duces a particularly powerful activator been identified. The SWI-SNFcomplex, activate transcription little when they by directly recruiting the holoenzyme which has recently been proposed to b~ are placed alone in a promoter, but to the target promoter in the absence o[ associated with the holoenzyme3s, is a greatly accentuate the activation of ad- a classical activation domain. We de- prime candidate for contributing such jacent enhancing sequences, presumably cided, therefore, to examine if recruiting an activity. Several in vitro studies using by providing a chromatin environment the holoenzyme to the PH05 promoter both yeast and human versions o[ this in which other activators can [unction through a Pho4 DNA-binding domain complex have shown that this complex could disrupt nucleosomes. To do so, can catalyse an ATP-dependent disordermore efficiently zo. [n the case of PH05, we can obtain we replaced the Pho4-activation domain ing of nucleosome structure that facili.. chromatin opening in the absence of with different components o! the RNA tares binding of transactivators anc~ transcription by mutating the TATAbox, polymerase II holoenzyme. Hybrid pro- TATA-binding protein3~8. Interestingly, as described above. However, we have teins hearing the DNA-bindingregion of nucleosome disrupticn at the PH05 pronot found a Pho4 derivative or mutant Pho4 fused to Gall 1 or Srb2 can remodel moter On a phoSO~' strain) requires that can open chromatin without acti- chromatin at the PH05 promoter, even glucose, but no other component of the vating transcription, suggesting that the with a TATA-deletedpromoter that can- growth medium, while nucleosome rechromatin reorganization activity of Pho4 not support transcription33. Our results formation proceeds in the absence of
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of a PH05 gene copy integrated into the URA3 locus, while the endogenous Pt105 Pho. gene remained repressed 4~. This indicates that the SIN4 gene product could be involved in insulating a promoter from its chromosomal environment. Consistent with this interpretation, we find that certain foreign DNA sequences in~UASpl ~ Pho4 Aact serted in the vicinity of the endogenous PH05 gene alleviate PH05 repression at high phosphate in a sin4 (but not in a wild-type) background, concomitant with a loss of nucleosome positioning over the promoter. Histone acetylaUon has been proAUASpl Pho4 Aact posed by many in the chromatin field to play a role in making nucleosomes more accessible to factor binding. This past year has marked a milestone in the chromatin field in that the elusive proteins that are responsible for histone acetylation and deacetylation have begun to be identified, as well as the protein Pho4 binding to UAS elements at the PH05 promoter. On the left, binding of Pho4 in vivo motifs that catalyse these processe~. (a) to the wild-type PH05 promoter and (b, ¢} to a variant promoter lacking UASpl is Satisfyingly, Gcn5, the protein that shown. (¢) is under conditions of Pno4 overproduction. On the right, in vivo binding of catalyses histone acetytatio# ?, was prePho4 (d) with or (e, f) without the activation domain, depicted as an oval, to the two UAS viously identified as a component of a elements at the PH05 promoter in their (d. e) natural and (f) reverse orientation is shown. coactivator complex required for the full The green arrow denotes initiation of transcription. activation potential of acidic activation domains4s. Conversely, a protein that glucose, suggesting that the disruption to attain true phosphate starvation con- had been identified as playing a role in of the nucleosomes is ATP-dependent 2~. ditions. Any mutation impairing growth silencing, Rpd3, has now been identiit was originally reported that a snt2 can therefore interfere with PH05 de- fied as a histone deacetylase49. Several mutation prevented activation of the repression without being directly in- years ago, it was shown that in an rpd3 PH05 gene39. We have found, however, volved in the activation of the PHO5 pro- mutation, neither lull repression nor that a deletion of the SNF2 gene, which motet. For tMs reason, we pregrow ceils full derepression of the PH05 gene is in other experiments has been shown under high phosphate conditions and achieved, leading to a diminished amplito disable SWI-,SNFfunction, has only a then shift them into a synthetic medium tude of regulation'% Defining the preminor effect on PHO5 activation and lacking phosphate altogether, rather cise role of such chromatln modifiers in chromatin disruption at the PH05 pro- than using low phosphate medium. PH05 regulation will further refine our moter~. However, there are several picture of how chromatin structure condistinct activities capable of labilizing Novel m ~ l a t m s of the PH05 trois gene activation. histone-DNAinteractions, and they might Evidence that the SWJ-SNF complex prove to be components of the holo- functions through chromatin structure Conclusions enzyme or at least transiently associate came originally from the isolation of It has become abundantly clear that with it. An example of such an activity bypass suppressor mutations called sin nucleosome structure is not merely an from Drosophila (NURF, nucleosome- (switch independent). The sinl suppres- ancestral packing material, but instead remodeling factor) 4°does not contain the sor mutant lies in a gene that bears is a dynamic structure that is exploited Drosophila homologue of Snf2, brahma, some similarity to HMG-I (Ref. 42), an by the cell for complex differential regubut instead contains another member of abundant chromosomal protein. The sin2 lation of gene activity. A corollary of this the Snf2 family, called iswi41. Given the suppressor is a mutant in one of the idea is that the nucleosome is not a number of Snf2-related proteins in yeast two copies of the histone H3 gene 43. constant, but rather can play a number identified by the yeast genome project, One interesting member of the Sin family of roles in gene regulation depending there could be a family of such activities of regulators is the sin4 suppressor mu- on its structure, modification state and that might play at least partially redun- tation~. The phenotype of this mutant chromosomal context. Our work on dant roles. is similar to yeast in which histone syn- PH05 indicates that chromatin has a reThe seemingly contradictory data on thesis is inhibited (see above). First, sev- pressive effect on th ,~promoter and that the role of Sn[2 in PH05 regulation eral promoters lacking UAS elements are chromatin opening is an integral part of might have to do with the fact that the activated in this mutant strai# 5. Second, the transcriptional activation process. A low phosphate medium, which is often the superhelical densities of yeast play regulatory role for chromatin is corrobouse(i to induce the PHO system, con- mids are decreased, which could indicate rated by a wealth of genetic evidence tains phosphate in concentrations high inhibition of nucleosome assembly44. It showing that the growing list of factors enough to repress PH05, and phosphate was recently reported that a sin4 mutation that regulate transcription includes chromust be consumed by growth of a culture leads to Pho4-independent derepression matin components (histories and HMG wi PHO5 pmmotor ~
(a) ~ - ~ - -
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(d) ----oP~--.--,-----~ ~ ~ p~
Rgure3
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proteins) as well as proteins that modify chromatin (acetylases and deacetylases). The recent completion 9f the yeast genome sequence has aided ia the idefltification of p r o t e i n s that c o u l d potentially modify chromatin structure, and efforts in this field will become inc r e a s i n g l y ~ocused on how these proteins determine promoter activity.
Aok~ow~e~e[ae~ts This work was supported by the Deutsche Forschungsgeme~nschaft (Sl~ 199) and Fends der Chemischen lndustrie. RefereJlces I Vogel, K.and Hinnen, A. (1990) Mol. Microbial. 4, 2013-2017 2 Ve~ter, U. a~d Horz. W. (1989) Nucleic Acids Res. 17, 1353-1369 3 Koren. R., LeVitre, J. and Bostian, K. A. (1986) Gene 41,271-280 4 8erben, G., Legrain, M., Gilliquet, V. and Hilger, F. (1990) Yeast 6,451--454 5 Sengstag, C. and Hinnen, A. (1987) Nucleic Acids Res. 15, 233-246 6 Berben, G., Legrain, M. and Hilger, F. (1988) Gene 66, 307-312 7 Vogel K., Hdrz, W. and Hinnen, A. (1989) MoL Celt, Biol. 9, 2050-2057 8 Rudolph, H. and Hinnen, A. (1987) Prec. Natl. Acad. Sci. U. S. A. 84, 1340-1344 9 Barbaric, S.. MSnsterkdtte~, M,, Svaren, J. and H~rz, W. (1996) Nucleic Acids Res. 24,
4479-4486 10 Hirst, K., Fisher, F., McAndrew, P. C. and Goding, C. R. (19941 EMBO 3. 13, 5450-5420 I 1 6razas, R. M and Stillmat~, D. J. (1993) Mol. Celt. Biol. 3.3, 5524-5537, correction 7200 12 Kaffman, A.. Herskowitz, I., Tjian, R. and O'Shea, E. K. (1994) Scie~Jce 263, 1153-1156 13 Lenburg, M. E. and O'Shea, E. K. (1996) Trends Biocl~em. Sci. 21, 383-387 14 Almer, A. and Hd/z, W. (1986) EMBO J. 5,
2881- 2687 15 Almer, A., Rudolph, H., Hinnen, A. and H6rz, W. (!986} EMBO J. 5, 2689~--2696 16 Hayes, J. J. and Wolffe, A, P. (1992) Prec. Natl. Acad. Sci. U. S. A. 89, 1229-1233 17 Straka, C. and H6rz, W. (1991) EMBOJ. 10. 361-368 18 Han, M., Kim, U. £, Kayne, P. and Grunstein, M. (1988} EMBO ./. 7, 2221-2228 19 Han, M. and Grunstein, M. (1988) Cell 55, 1137-1145 20 Venter, U. et at. (1994) EMBO J. 13, 4848-4855 21 Svaren, J., Schmitz, J. and H6rz, W. (1994) EMBO J. 13, 4856-4862 22 Pedmann, T. and Wrange, O. (1988) EMBO J. 7, 3073-3079 23 Adams, C. C. and Workman, J. L. (1993) Cell 72, 305-308 24 Barbaric, S., Fascher, K. D. and H6rz, W. (1992) Nucleic Acids Res. 20, 1031-1038 25 Schmid, A., Fascher, K. D. and H6rz, W. (1992) Celt 71, 853-864 26 Fascher, K. D., Schmitz, J. and H6rz, W. (1993) J. MoL Biol. 231, 658-667 27 Sengstag. C. and Hinnen, A. (1988) Gene 67, 223--228 28 Axelrod, J. D., Reagan, M. S. and Majors, J. (1993) Genes Dev. 7,857-869
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derivatives) that were shorter than the sum of the van der Waals radii of the atoms involved. Furthermore, in 1982, a survey of numerous neutron structures conclusively established the existence 9f C-H,,.Y hydrogen bonds4. The strongest hydrogen bond is the C-H.,oO contact, which exhibits typical directional and electrostatic features (Fig. 1). C-H°°.O bond strengths often correlate with the Observations of short C-H...O contacts in biological macromolecules, includacidity of the hydrogen attached to the ing nucleic acids, proteins and carbohydrates, suggest that ttlese unconvencarbon atom, with methyne and methyltional hydrogen bonds have both a structurally and functionally important role. ene groups being stronger donors than methyl groupss. Carbons with adjacent HYDROGEN BONDS, ARE one of the aqueous environment and ligands. They nitrogen atoms form particularly strong most important types of interactions in are attractive electrostatic interactions hydrogen bonds4. High-precision neutron molecular biology, shaping not only of the type X-H°°oY, in which Y, the diffraction structures and theoretical the three-dimensional structures of hydrogen-bond acceptor, carries a full studies indicate that the covalent C-H macromolecules, but also influencing or partial negative charge, while the bond lengthens when the hydrogen atom their interactions with the surrounding hydrogen-bond donor, X, is more negative is involved in a hydrogen bond contact~ . The surface of macromolecules conthan H. To maximize the electrostatic attraction, the hydrogen preferentially sists mainly of groups that can act either M, C, Wahl is at the Max-Planck-lnstitut for approaches Y along the direction of and as donors or as acceptors of C-H°o.O/N Biochemie, Abteilung Strukturforschung, in a plane with a lone-pair orbital of Y hydrogen bonds. Until recently, many Am Klopferspitz 18a, 1:)432512 PlaneggMartinsried, Germany; and M. Sundarallngam (see Ref. 1 for a review). Sutor first sum- cases of C-Ho..O interactions were classiis at the Laboratory of Biological marized the crystallographic evidence fied as hydrophobic interactions and Macromolecular Structure, Departments of in support of the existence of C-H...O not properly identified. Owing to the Chemistry and Biochemistry, The Ohio State hydrogen bonds2,3. She observed that l ~ e size of the macromolecules and the University, 120 West 18th Avenue Columbus, there were contacts in some crystal limited resolution, X-ray studies cannot OH 43210-1002, USA. structures (of nucleic acid bases and locate hydrogen atoms. While C-H.o°O Email: [email protected]
C=H.,oOhy&'ogenbondingin biology Markus C. Wahl and MuttaiyaSundaralingam
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