A 16-residue peptide [(Ala-Glu-Ala-Glu-AlaLys-Ala-Lys)2J has a characteristic 13-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes.Scanning EM reveals a network of interwoven framents ""10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.Peptides of alternating hydrophilic and hydrophobic amino acid residues have a tendency to adopt a (3-sheet structure. The complete sequence of (Ala-Glu-Ala-Glu-Ala-Lys-AlaLys)2 (EAK16) was originally found in a region of alternating hydrophobic and hydrophilic residues in zuotin, a yeast protein that was initially identified for its ability to bind preferentially to left-handed Z-DNA (1). Previous studies with alternating amphiphilic-peptide polymers-e.g., poly-(Val-Lys), poly(Glu-Ala), poly(Tyr-Glu), poly(Lys-Phe), poly(Lys-Leu)-and oligopeptides [(Val-Glu-Val-Orn)j_3]-Val (2-7) have shown that these polymers can adopt (3-sheet structures and can aggregate, depending upon pH, salt, and time. However, self-complementary EAK16 is distinctive in that it forms an insoluble macroscopic membrane. MATERIALS AND METHODSPeptides. The Glu-Ala-Lys peptides were synthesized by a peptide synthesizer (Applied Biosystems), purified by reverse-phase HPLC, and eluted by a linear gradient of 5-80% acetonitrile/0.1% trifluoacetic acid. The peptide stock solutions were dissolved in water (1-5 mg/ml) or in 23% acetonitrile (10 mg/ml). The concentrations of the peptides were determined by dissolving dried peptide in water (wt/vol) and centrifuging the solution. A portion of the solution was then analyzed by hydrolysis with internal controls. The sequence of the peptides was confirmed by microsequencing. The composition of the peptides was confwrmed by hydrolytic analysis. Ala-Glu-Ala-Lys-Ala-Glu-Ala-Glu-Ala-Lys-AlaLys (EAK12) and EAK16 are acetylated and aminated at the N-and C-terminal ends, respectively. Blocking of both N and C termini of EAK16 appears nonessential for membrane formation.CD Measurement. CD spectra were gathered on an Aviv model 6ODS spectropolarimeter with 6OHDS software for data processing. Because EAK16 contains both positively and negatively charged residues, the peptide itself can serve as a buffer. CD samples were prepared by diluting stock peptide solution (1-5 mg/ml) in water.Membrane Preparations. The membranes were prepared as follows: 5-10 ,4 of the stock solution of EAK16 peptide (1-5 mg/ml) was added to 0.5-1.0 ml of phosphate-buffered saline (150 mM NaCl/10 mM sodium phosphate, pH 7.4) with 0.00001% Con...

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH2) is able to form an unusually stable beta-sheet structure. The beta-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the beta-sheet structure. The beta-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide beta-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of beta-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research.

A putative Z‐DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z‐DNA binding assay using [32P]poly(dG‐m5dC) and [32P]oligo(dG‐Br5dC)22 in the presence of B‐DNA competitor. Poly(dG‐Br5dC) in the Z‐form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG‐dC) and poly(dA‐dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG‐dC)7 segment in the Z‐form was an excellent competitor. A Southwestern blot using [32P]poly(dG‐m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N‐terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z‐DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

The crystal structure of d(C3T), solved at 1.4 A resolution, reveals that the molecule forms a four-stranded intercalated complex. It consists of two parallel-stranded duplexes, each of which Is held together by cytosine-protonated cytosine base pairs. The two duplexes are intercalated with each other and have opposite strand orientation. The molecule has a flat, lath-like appearance, and the covalently bonded cytosines have a slow right-handed twist of 17.1°. However, there is considerable asymmetry. On one of the flat sides, the phosphate groups are rotated away from the center of the molecule. They are held In this orientation by bridging water molecules that bind the NH of cytosine and a phosphate group of an opposite chain. There is also considerable microheterogeneity in the structure. The cytosine hemiprotonation occurs even at pH 7 where stable crystals form.For some time it has been known that nucleic acids containing stretches of cytidine residues can form parallel strands held together by cytosine-protonated cytosine base pairs (C-C+) (1-4). In an NMR analysis of d(TC5), Gudron and his associates (5) proposed an unusual structure in which two such parallel-stranded duplexes, held together by C'C+ base pairs, intercalate with each other in opposite polarity to form a four-stranded molecule. The evidence for the structure was based on strong interactions between the Hi' protons on different strands, suggesting that the backbones were close together. Recent crystal structures of d(TAACCC) (C.K., I.B., C.L., R.R., R.M., and A.R.), (unpublished data) and of d(C4) (6) were in agreement with the general conclusions deduced by the NMR studies. In telomeres at the end of chromosomes, stretches of cytosine residues are found, usually linked to thymine residues. Here, we present a crystallographic analysis of the structure of d (C3T) Rotation searches of all the models indicated a molecule with the helical axis approximately parallel to the crystallographic b axis, as we expected. Subsequent translation searches of some of the models yielded positions for the molecule that were free of close van der Waals contacts. Rigid body refinement followed by intensive simulated annealing using the program XPLOR (7) improved the density around the phosphate groups ofthe backbone and allowed us to build another model with more accurate helical twist based on the phosphate positions. We used this improved molecule as a starting model and molecular replacement was performed again. From the very beginning, we could identify two thymine residues stacked on top of six C-C+ layers and added those to the model. At this stage, simulated annealed omit maps (6) clearly showed the location of the remaining two thymine residues stacked perpendicular to the helix axis. Positional refinement followed by refinement of the temperature factors led to a final R factor of 17.7% for 5013 reflections above the 2cr level (based on F.) between 10 and 1.4 A. The free R factor (8) value based on a random 10%o subset of reflections is 22.5%...

BiochemistryStable loop in the crystal structure of the intercalated four-stranded cytosine-rich metazoan telomere (C-C+ ABSTRACTIn most metazoans, the telomeric cytosinerich strand repeating sequence is d(TAACCC). The crystal structure of this sequence was solved to 1.9-A resolution. Four strands associate via the cytosine-containing parts to form a four-stranded intercalated structure held together by C C+ hydrogen bonds. The base-paired strands are parallel to each other, and the two duplexes are intercalated into each other in opposite orientations. One TAA end forms a highly stabilized loop with the 5' thymine Hoogsteen-base-paired to the third adenine. The 5' end of this loop is in close proximity to the 3' end of one of the other intercalated cytosine strands. Instead of being entirely in a DNA duplex, this structure suggests the possibility ofan alternative conformation for the cytosine-rich telomere strands.

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediateearly promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IEl gene promoterenhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.Although human cytomegalovirus (HCMV) nonproductively infects cells of many types from humans (8,16,17) and other species (2, 11), the virus replicates in vitro only in fibroblasts of human origin. Transcription of the first viral genes expressed, the immediate-early (IE) genes, are mainly dependent on host cell factors (1, 5-7, 20, 21), suggesting that this may constitute the primary event at which expression of the viral genome is regulated. Regulatory sequences of the HCMV major IE gene, the IEl gene, encompass a polymerase II promoter between +1 and -65 (6, 19); a strong and complex enhancer between -66 and -524 (1, 5, 7), which is active in many different cell types; a cluster of nuclear factor 1-binding sites between -600 and -720 (9, 10); and modulator sequences between -750 and -1145 (14, 15). These modulator sequences negatively regulate expression in nonpermissive undifferentiated teratocarcinoma (Tera-2) cells but positively influence expression in permissive differentiated cells (14,15). In the present study we have addressed the question of whether the modulator region is functional in cell types other than the embryonal carcinoma cell system and whether indeed it may contribute to cellspecific expression.Recombinant plasmids containing the modulator (A1145) or with the modulator sequences truncated (A525), which were previously used to define the modulator region (15) A1145 and A525 in cells of various lineages were measured by scintillation counting of conversion rates from chloramphenicol to its acetylated forms. All cells were cotransfected with pRSV-luciferase as a transfection control (3), and the results with A1145 and A525 were adjusted according to the relative luciferase activity in the cell extracts. The effect of the modulator sequences on CAT activity differed depending on cell type (Fig. 1). A twofold negative effect was observed in H9, U937, and SW480 cells, but expression in Jurkat, 293, Raji, and Namalwa cells was barely affected. In B cells the expression was augmented about three-to fourfold by the upstream sequences. These results clearly indicate that the HCMV modulator sequences can influence IE promoterenhancer activity in vivo in cell types other than teratocarcinoma cells. In particular, the cis-acting sequences negatively modulate expression in some T cells and rectal epithelial cells, in which HCMV is found during natural infection...

The translational regulator protein regA is encoded by the T4 bacteriophage and binds to a region of messenger RNA (mRNA) that includes the initiator codon. RegA is unusual in that it represses the translation of about 35 early T4 mRNAs but does not affect nearly 200 other mRNAs. The crystal structure of regA was determined at 1.9 A resolution; the protein was shown to have an alpha-helical core and two regions with antiparallel beta sheets. One of these beta sheets has four antiparallel strands and has some sequence homology to RNP-1 and RNP-2, which are believed to be RNA-binding motifs and are found in a number of known RNA-binding proteins. Structurally guided mutants may help to uncover the basis for this variety of RNA interaction.

In this paper we demonstrate that modulator sequences upstream of the enhancer of the major immediate-early promoter of human cytomegalovirus exert a differential effect on the level of transcription in a variety of cells and that this region has the capacity to interact with specific nuclear protein. Depending on the cell type, these modulator sequences increased or decreased transcriptional activation from the IE1 gene promoter-enhancer. The cell lines identified in this report should be useful to study the molecular mechanism of cell-specific transcriptional repression and activation exerted by the major immediate-early promoter upstream region.