Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.

Binding site size limit of the 2:1 pyrrole-imidazole polyamide-DNA motif.

Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids can be combined in antiparallel side-by-side dimeric complexes for sequence-specific recognition in the minor groove of DNA. Six polyamides containing three to eight rings bind DNA sites 5-10 bp in length, respectively. Quantitative DNase I footprint titration experiments demonstrate that affinity maximizes and is similar at ring sizes of five, six, and seven. Sequence specificity decreases as the length of the polyamides increases beyond five rings. These results provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

Multivalent DNA-binding properties of the HMG-1 proteins.

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements.

Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.

Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations.

Physical and functional independency of p70 and p58 natural killer (NK) cell receptors for HLA class I: their role in the definition of different groups of alloreactive NK cell clones.

Natural killer (NK) cells express clonally distributed receptors for different groups of HLA class I alleles. The Z27 monoclonal antibody described in this study recognizes a p70 receptor specific for HLA-B alleles belonging to the Bw4 supertypic specificity. Single amino acid substitutions in the peptide-binding groove of HLA-B2705 molecules influenced the recognition by some, but not all, p7O/Z27+ clones. This suggests the existence of a limited polymorphism within the p7O family of receptors. The pattern of reactivity of monoclonal antibody Z27 revealed that Bw4-specific receptors may be expressed alone or in combination with different (GL183 and/or EB6) p58 molecules. Analysis of NK clones coexpressing p58 and p7O receptors allowed us to demonstrate that the two molecules represent physically and functionally independent receptors. The expression of p7O molecules either alone or in combination with EB6 molecules provided the molecular basis for understanding the cytolytic pattern of two previously defined groups of "alloreactive" NK cell clones ("group 3" and "group 5").

Sequence-selective carbohydrate-DNA interaction: dimeric and monomeric forms of the calicheamicin oligosaccharide interfere with transcription factor function.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide are known to bind to the minor groove of DNA in a sequence-selective manner preferring distinct target sequences. We tested these carbohydrates for their ability to interfere with transcription factor function. The oligosaccharides inhibit binding of transcription factors to DNA in a sequence-selective manner, probably by inducing a conformational change in DNA structure. They also interfere with transcription by polymerase II in vitro. The effective concentrations of the oligosaccharides for inhibition of transcription factor binding and for transcriptional inhibition are in the micromolar range. The dimer is a significantly more active inhibitor than is the monomer.

Crystal structure of the complex of a catalytic antibody Fab fragment with a transition state analog: structural similarities in esterase-like catalytic antibodies.

The x-ray structure of the complex of a catalytic antibody Fab fragment with a phosphonate transition-state analog has been determined. The antibody (CNJ206) catalyzes the hydrolysis of p-nitrophenyl esters with significant rate enhancement and substrate specificity. Comparison of this structure with that of the uncomplexed Fab fragment suggests hapten-induced conformational changes: the shape of the combining site changes from a shallow groove in the uncomplexed Fab to a deep pocket where the hapten is buried. Three hydrogen-bond donors appear to stabilize the charged phosphonate group of the hapten: two NH groups of the heavy (H) chain complementarity-determining region 3 (H3 CDR) polypeptide chain and the side-chain of histidine-H35 in the H chain (His-H35) in the H1 CDR. The combining site shows striking structural similarities to that of antibody 17E8, which also has esterase activity. Both catalytic antibody ("abzyme") structures suggest that oxyanion stabilization plays a significant role in their rate acceleration. Additional catalytic groups that improve efficiency are not necessarily induced by the eliciting hapten; these groups may occur because of the variability in the combining sites of different monoclonal antibodies that bind to the same hapten.

Evidence for DNA phosphate backbone alkylation and cleavage by pyrrolo[1,2-a]benzimidazoles: small molecules capable of causing base-pair-specific phosphodiester bond hydrolysis.

This report presents evidence that a reduced pyrrolo[1,2-a]benzimidazole (PBI) cleaves DNA as a result of phosphate alkylation followed by hydrolysis of the resulting phosphate triester. The base-pair specificity of the phosphate alkylation results from Hoogsteen-type hydrogen bonding of the reduced PBI in the major groove at only A.T and G.C base pairs. Alkylated phosphates were detected by 31P NMR and the cleavage products were detected by 1H NMR and HPLC. Evidence is also presented that a reduced PBI interacts with DNA in the major groove rather than in the minor groove or by intercalation.

Crystal structure of a DNA decamer showing a novel pseudo four-way helix-helix junction.

The crystal structure of the decanucleotide d(CGCAATTGCG)2 has been solved by a combination of molecular replacement and heavy-atom procedures and has been refined to an R factor of 20.2% at 2.7 A. It is not a fully base-paired duplex but has a central core of eight Watson-Crick base pairs flanked by unpaired terminal guanosines and cytosines. These participate in hydrogen-bonding arrangements with adjacent decamer duplexes in the crystal lattice. The unpaired guanosines are bound in the G+C regions of duplex minor grooves. The cytosines have relatively high mobility, even though they are constrained to be in one region where they are involved in base-paired triplets with G.C base pairs. The 5'-AATT sequence in the duplex region has a narrow minor groove, providing further confirmation of the sequence-dependent nature of groove width.

Similar antigenic surfaces, rather than sequence homology, dictate T-cell epitope molecular mimicry.

Molecular mimicry, normally defined by the level of primary-sequence similarities between self and foreign antigens, has been considered a key element in the pathogenesis of autoimmunity. Here we describe an example of molecular mimicry between two overlapping peptides within a single self-antigen, both of which are recognized by the same human self-reactive T-cell clone. Two intervening peptides did not stimulate the T-cell clone, even though they share nine amino acids with the stimulatory peptides. Molecular modeling of major histocompatibility complex class II-peptide complexes suggests that both of the recognized peptides generate similar antigenic surfaces, although these are composed of different sets of amino acids. The molecular modeling of a peptide shifted one residue from the stimulatory peptide, which was recognized in the context of the same HLA molecule by another T-cell clone, generated a completely different antigenic surface. Functional studies using truncated peptides confirmed that the anchor residues of the two "mimicking" epitopes in the HLA groove differ. Our results show, for two natural epitopes, how molecular mimicry can occur and suggest that studies of potential antigenic surfaces, rather than sequence similarity, are necessary for analyzing suspected peptide mimicry.

Direct observation of disordered regions in the major histocompatibility complex class II-associated invariant chain.

Invariant chain (Ii) is a trimeric membrane protein which binds and stabilizes major histocompatibility complex class II heterodimers in the endoplasmic reticulum and lysosomal compartments of antigen-presenting cells. In concert with an intracellular class II-like molecule, HLA-DM, Ii seems to facilitate loading of conventional class II molecules with peptides before transport of the class II-peptide complex to the cell surface for recognition by T cells. The interaction of Ii with class II molecules is thought to be mediated in large part through a region of 24 amino acids (the class II-associated Ii peptide, CLIP) which binds as a cleaved moiety in the antigenic peptide-binding groove of class II molecules in HLA-DM-deficient cell lines. Here we use nuclear magnetic resonance techniques to demonstrate that a soluble recombinant Ii ectodomain contains significant disordered regions which probably include CLIP.

Structural features of the invariant chain fragment CLIP controlling rapid release from HLA-DR molecules and inhibition of peptide binding.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

A molecular anchor for stabilizing triple-helical DNA.

Molecular modeling has been used to predict that 2,6-disubstituted amidoanthraquinones, and not the 1,4 series, should preferentially interact with and stabilize triple-stranded DNA structures over duplex DNA. This is due to marked differences in the nature of chromophore-base stacking and groove accessibility for the two series. A DNA foot-printing method that monitors the extent of protection from DNase I cleavage on triplex formation has been used to examine the effects of a number of synthetic isomer compounds in the 1,4 and 2,6 series. The experimental results are in accord with the predicted behavior and confirm that the 1,4 series bind preferentially to double- rather than triple-stranded DNA, whereas the isomeric 2,6 derivatives markedly favor binding to triplex DNA.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

CC-1065 and the duocarmycins: unraveling the keys to a new class of naturally derived DNA alkylating agents.

Key studies defining the DNA alkylation properties and selectivity of a new class of exceptionally potent, naturally occurring antitumor antibiotics including CC-1065, duocarmycin A, and duocarmycin SA are reviewed. Recent studies conducted with synthetic agents containing deep-seated structural changes and the unnatural enantiomers of the natural products and related analogs have defined the structural basis for the sequence-selective alkylation of duplex DNA and fundamental relationships between chemical structure, functional reactivity, and biological properties. The agents undergo a reversible, stereoelectronically controlled adenine-N3 addition to the least substituted carbon of the activated cyclopropane within selected AT-rich sites. The preferential AT-rich non-covalent binding selectivity of the agents within the narrower, deeper AT-rich minor groove and the steric accessibility to the alkylation site that accompanies deep AT-rich minor groove penetration control the sequence-selective DNA alkylation reaction and stabilize the resulting adduct. For the agents that possess sufficient reactivity to alkylate DNA, a direct relationship between chemical or functional stability and biological potency has been defined.