Optimal Binding Site Research Articles

Sex-lethal Interactions with Protein and RNA: ROLES OF GLYCINE-RICH AND RNA BINDING DOMAINS

Sex-lethal (Sxl) is an RNA-binding protein, containing two conserved RNA binding domains (RBDs) and a glycine-rich region, which functions as a regulator of alternative splicing in Drosophila sex determination. Previous work demonstrated that Sxl monomers interact cooperatively upon binding to target RNAs and that the cooperativity depends on the glycine-rich N terminus. Here we use band shift experiments to show that RNA binding patterns are altered when Sxl is combined with other proteins having similar glycine-rich domains, including mammalian heterogeneous nuclear (hn) RNP L and Drosophila Hrb87F (an hnRNP A/B homolog). Direct involvement of the Sxl glycine-rich region in protein interactions was verified by Far-Western analysis. Two interaction domains, the Sxl N terminus and the Sxl first RNA binding domain, were suggested by the yeast two-hybrid assay. In a systematic examination of the RNA binding properties of Sxl domains, it was found that the Sxl termini as well as the RBDs influence RNA binding specificity. Finally, selection of the Sxl optimal binding site (SELEX) confirms the importance of U-runs in the Sxl binding site and suggests a second type of non-U-run target that may be associated with RNA secondary structure.

Engrailed (Gln50-->Lys) homeodomain-DNA complex at 1.9 A resolution: structural basis for enhanced affinity and altered specificity.

The homeodomain is one of the key DNA-binding motifs used in eukaryotic gene regulation, and homeodomain proteins play critical roles in development. The residue at position 50 of many homeodomains appears to determine the differential DNA-binding specificity, helping to distinguish among binding sites of the form TAATNN. However, the precise role(s) of residue 50 in the differential recognition of alternative sites has not been clear. None of the previously determined structures of homeodomain-DNA complexes has shown evidence for a stable hydrogen bond between residue 50 and a base, and there has been much discussion, based in part on NMR studies, about the potential importance of water-mediated contacts. This study was initiated to help clarify some of these issues. The crystal structure of a complex containing the engrailed Gln50-->Lys variant (QK50) with its optimal binding site TAATCC (versus TAATTA for the wild-type protein) has been determined at 1.9 A resolution. The overall structure of the QK50 variant is very similar to that of the wild-type complex, but the sidechain of Lys50 projects directly into the major groove and makes several hydrogen bonds to the O6 and N7 atoms of the guanines at base pairs 5 and 6. Lys50 also makes an additional water-mediated contact with the guanine at base pair 5 and has an alternative conformation that allows a hydrogen bond with the O4 of the thymine at base pair 4. The structural context provided by the folding and docking of the engrailed homeodomain allows Lys50 to make remarkably favorable contacts with the guanines at base pairs 5 and 6 of the binding site. Although many different residues occur at position 50 in different homeodomains, and although numerous position 50 variants have been constructed, the most striking examples of altered specificity usually involve introducing or removing a lysine sidechain from position 50. This high-resolution structure also confirms the critical role of Asn51 in homeodomain-DNA recognition and further clarifies the roles of water molecules near residues 50 and 51.

The winged-helix transcription factor Trident is expressed in cycling cells.

We describe the cloning and characterization of Trident , a novel member of the fork head/winged-helix family, from murine thymus. In the mouse embryo, the gene was expressed in all tissues, whereas in adult mice expression was only detected in the thymus. Further analysis revealed that Trident expression strictly correlated with cell cycling, independent of cell type. Timing of [3H]thymidine incorporation showed that mRNA and protein expression were strongly upregulated upon entry into the S phase of the cell cycle. Moreover, the protein was phosphorylated in M phase. PCR-mediated selection of optimal binding sites yielded a consensus motif resembling that of other family members. These results identify Trident as a transcription factor, which is likely involved in cell cycle-specific gene regulation.

An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

Characterization of a zinc-dependent transcriptional activator from Arabidopsis.

The C2-H2 zinc-finger is a widely occurring DNA binding motif, usually present as tandem repeats. The majority of C2-H2 zinc-finger proteins that have been studied are derived from animals. Here, we characterize a member of a distinct class of plant C2-H2 zinc-finger proteins in detail. A cDNA clone encoding a DNA binding protein from Arabidopsis was isolated by SouthWestern screening. The protein, termed ZAP1 (Zinc-dependent Activator Protein-1), is encoded by a single copy gene, which is expressed to similar levels in root and flower, to a somewhat lower level in stem and to low levels in leaf and siliques. The optimal binding site was determined by random binding site selection, and the consensus sequence found is CGTTGACCGAG. The homology between ZAP1 and other DNA binding proteins is restricted to a repeated region of a stretch of 24 highly conserved amino acids followed by a zinc-finger motif (C-X4-C-X22-23-H-X1-H). The C-terminal zinc-finger region is essential for DNA binding, whereas deletion of the N-terminal one resulted in 2.5-fold reduced binding affinity. Binding of ZAP1 to DNA was abolished by metal-chelating agents. The activation domain as determined in yeast is adjacent to and possibly overlapping with the DNA binding domain. Particle bombardment experiments with plant cells showed that ZAP1 increases expression of a gusA reporter gene that is under control of ZAP1 binding sites. We conclude that ZAP1 is a plant transcriptional activator with a C2-H2 zinc-finger DNA binding domain.

Identification of Optimal Binding Sites for PPAR/RXR Heterodimers

Identification of Optimal Binding Sites for PPAR/RXR Heterodimers

Novel DNA binding specificities of a putative herpesvirus bZIP oncoprotein.

Marek's disease virus is a highly oncogenic herpesvirus that can cause T lymphomas and peripheral nerve demyelination in chickens. meq, a candidate oncogene of Marek's disease virus, encodes a basic leucine zipper (bZIP) transcription factor which contains a large proline-rich domain in its C terminus. On the basis of its bZIP structural homology, meq is perhaps the only member of the jun-fos gene family completely viral in origin. We previously showed that Meq's C-terminal domain has potent transactivation activity and that its bZIP domain can dimerize with itself and with c-Jun also. In an effort to identify viral and cellular targets of Meq, we have determined the optimal binding sites for Meq-Jun heterodimers and Meq-Meq homodimers. By a PCR-based approach using cyclic amplification of selected targets, Meq-Jun heterodimers were found to optimally bind tetradecanoylphorbol acetate response element (TRE) and cyclic AMP response element (CRE) consensus sequences. This result was consistent with the results of our previous functional analysis implicating Meq-Jun heterodimers in the transactivation of the Meq promoter through a TRE- or CRE-like sequence. Interestingly, Meq-Meq homodimers were found to bind two distinct motif elements. The first [GAGTGATG AC(G)TCATC] has a consensus which includes a TRE or CRE core flanked by additional nucleotides critical for tight binding. Methylation interference and mutational analyses confirmed the importance of the flanking residues. The sequences of a subset of TRE and CRE sites selected by Meq-Meq are closely related to the binding motif of Maf, another bZIP oncoprotein. The second putative Meq binding site (RACACACAY) bears a completely different consensus not shared by other bZIP proteins. Binding to this consensus sequence also requires secondary structure characteristics associated with DNA bending. CACA motifs are known to promote DNA curvature and function in a number of special biological processes. Our results lend further weight to the increasing importance of DNA bending in transcriptional regulation and provide a baseline for the identification of Meq-responsive targets.

Characterisation of silica-based heparin-affinity adsorbents through column chromatography of plasma fractions containing thrombin

Different heparin-silicas, synthesised in this laboratory via directional end-point attachment of heparin (H) onto various amino-derivatised silicas, have been evaluated in packed-bed and expanded-bed column chromatographic experiments using crude preparations of the therapeutic protein, thrombin (T). Adsorbent capacities, determined through batch adsorption experiments, were verified by employing frontal analysis in packed-bed systems. The performance of these adsorbents was also investigated in terms of thrombin purification factors and recoveries. The potential of the heparin-silicas was further examined in the expanded-bed column chromatographic mode using a scaled-up procedure. With heparin-Fractosil 1000 adsorbents, capacities of around 100 000 U thrombin/ml adsorbent could be achieved. Heparin-Fractosil 1000 adsorbents of intermediate heparin content (around 4 mg heparin/ml sorbent) displayed binding stoichiometries similar to that of the commercial heparin-Sepharose (2.6–2.7 mol T/mol H). Furthermore, binding stoichiometries were largely unaffected by increasing the heparin content on the heparin-Fractosil 1000 adsorbents from 0.8 to 4.6 mg of heparin/ml of sorbent. This result suggests that optimal binding site accessibilities for the thrombin-heparin interaction occurs at lower ligand density values. The binding capacity values determined from frontal analysis were confirmed by the recovery data, thus indicating minimal irreversible adsorption. Specific activities of ca. 2100 U/mg were obtained for thrombin when affinity-purified on these heparin-LiChroprep Si60 or heparin-Fractosil 1000 adsorbents. These values were higher than the maximum achievable purity obtained through alternative, multi-step chromatographic purification procedures reported by other investigators. These results indicated that the packed-bed performances with these silica-based adsorbents were superior to currently available commercial soft gel adsorbents, with the more dense heparin-silicas exhibiting very good potential for use in expanded-bed applications.

Characterization of the dead ringer Gene Identifies a Novel, Highly Conserved Family of Sequence-Specific DNA-Binding Proteins

We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.

Characterization of the Hypoxia-inducible Protein Binding Site within the Pyrimidine-rich Tract in the 3′-Untranslated Region of the Tyrosine Hydroxylase mRNA

Reduced tension of O2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552 1578 in the 3 -untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia-induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species.

DNA binding through distinct domains of zinc-finger-homeodomain protein AREB6 has different effects on gene transcription.

Transcription factor AREB6 has a unique structure composed of two zinc-finger clusters in N- and C-terminal regions, and one homeodomain in the middle. AREB6 has been known to regulate the expression of the Na, K-ATPase alpha 1 subunit, interleukin 2 and delta-crystallin genes. We determined the optimal binding sites for the N-terminal zinc-finger cluster as GTCACCTGT or TGCACCTGT and for the C-terminal zinc-finger cluster as C/TACCTG/TT by the CASTing method (cyclic amplification and selection of targets). The additional consensus sequence GTTTC/G, in conjunction with the CACCTGT sequence, was selected by the second CASTing for the entire coding region. The N-terminal zinc-finger cluster binds to DNA strongly when the DNA has GTTTC/G in conjunction with the CACCTGT sequence. The homeodomain had no specific DNA binding activity but was found to interact with the N-terminal zinc-finger cluster. Analyses of zinc-finger mutation proteins revealed that the contribution to DNA binding of each N-terminal zinc-finger motif is altered depending on the presence of the additional consensus. Transient transfection assays showed that AREB6 repressed the human 70-kDa heat-shock gene promoter harboring the CACCTGT sequence together with the additional consensus, and that AREB6 activated the promoter harboring the CACCTGT sequence without the additional consensus. These results suggest that AREB6 has multiple conformational states, leading to positive and negative regulations of gene transcription.

Operator binding by lambda repressor heterodimers with one or two N-terminal arms.

The first 6 amino acids (NH2-Ser1-Thr2-Lys3-Lys4-Lys5-Pro6) of bacteriophage lambda cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric lambda repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of lambda repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain-operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons

A search for POU domain sequences expressed in the human retina has led to the identification of three closely related genes: Brn-3a, Brn-3b, and Brn-3c. The structure and expression pattern of Brn-3b was reported earlier (Xiang et al., 1993); we report here the structures and expression patterns of Brn-3a and Brn-3c. Antibodies specific for each Brn-3 protein were generated and shown to label only ganglion cells in a variety of vertebrate retinas. A complex pattern of strongly and weakly immunolabeled ganglion cells was observed in mouse, cat, and monkey retinae. In mouse and cat retinae, Brn-3a and Brn-3b proteins are found in a large fraction of ganglion cells, whereas Brn-3c is present in fewer ganglion cells. In the cat retina, anti-Brn-3a immunoreactivity was strong in the small ganglion cells (gamma cells) and weak in the remaining ganglion cells (alpha and beta cells); anti-Brn-3b immunoreactivity was present in all ganglion cells; and anti-Brn3c immunoreactivity was confined to the small ganglion cells. Immunolabeling of macaque retinae following retrograde labeling from the lateral geniculate nucleus revealed strong anti-Brn-3a immunoreactivity in a minority of retrogradely labeled P-type ganglion cells, and weak Brn-3a immunoreactivity in all of the remaining P- and M-type ganglion cells. In the same retinae, strong anti-Brn-3b immunoreactivity was seen in nearly all P-type ganglion cells and weak immunoreactivity in nearly all M-type ganglion cells. Each of the Brn-3-specific antibodies also labeled subsets of neurons in the dorsal root and trigeminal ganglia, suggesting that primary somatosensory neurons and retinal ganglion cells share genetic regulatory hierarchies. In vitro selection of an optimal DNA binding site using the Brn-3b POU domain has revealed a consensus [(A/G)CTCATTAA(T/C)] that is recognized by each of the Brn-3 POU domains and is distinct from binding sites previously described for other POU domain proteins.

EBF contains a novel zinc coordination motif and multiple dimerization and transcriptional activation domains.

Early B cell factor (EBF) was identified and cloned as a transcription factor expressed specifically in B lymphocytes and adipocytes. This protein was also identified as olfactory factor 1 (Olf-1) in olfactory neurons. In this study, we analyzed the structural requirements for DNA binding, homodimerization and transcriptional activation by EBF. A carboxyl-terminal region, containing a repeat of alpha-helices related to the helix-loop-helix motif, is important for dimerization of EBF in solution and can confer dimerization upon a heterologous DNA binding protein. The amino-terminal DNA binding domain by itself is monomeric, but can mediate assembly of dimers on optimized and correctly spaced half-sites. Mutational analysis of the DNA binding domain of EBF indicated that a novel zinc coordination motif consisting of H-X3-C-X2-C-X5-C is important for DNA recognition. Deletion analysis and transfer of regions of EBF onto a heterologous DNA binding domain identified a serine/threonine-rich transcriptional activation domain. Moreover, the DNA binding domain of EBF can mediate transcriptional activation from optimized binding sites. Thus, EBF contains both a complex DNA binding domain that allows for dimerization and transcriptional activation, and additional dimerization and activation domains.

Components of a stat recognition code: Evidence for two layers of molecular selectivity

Latent and activated forms of Stat1 and Stat6 have been expressed and purified, enabling biochemical experiments relating to their functional activities. Stat1 bound to a phosphotyrosine peptide derived from the IFNγ receptor with a K D of 50 nM, whereas Stat6 bound to an IL-4 receptor peptide with a K D of 300 nM. Stat-receptor peptide interactions were specific and dependent upon tyrosine phosphorylation. Activated forms of Stat1 and Stat6 were used to select their optimal DNA binding sites. Stat1 selected a recognition site having dyad half-sites separated by 3 bp. Stat6 selected a recognition site composed of the same dyad half-sites, yet separated by 4 bp. Chimeric Stat1-Stat6 recombinants were expressed, purified, and assayed for receptor coupling and DNA binding specificity. Such studies led to the Identification of polypeptide domains that specify these activities. These observations provide a framework for understanding how different cytokines elicit distinctive patterns of gene expression.

AUUUA is not sufficient to promote poly(A) shortening and degradation of an mRNA: the functional sequence within AU-rich elements may be UUAUUUA(U/A)(U/A).

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

AUUUA Is Not Sufficient To Promote Poly(A) Shortening and Degradation of an mRNA: the Functional Sequence within AU-Rich Elements May Be UUAUUUA(U/A)(U/A)

AU-rich elements (AREs) in the 3' untranslated regions of several cytokine and oncogene mRNAs have been shown to function as signals for rapid mRNA degradation, and it is assumed that the many other cytokine and oncogene mRNAs that contain AU-rich sequences in the 3' untranslated region are similarly targeted for rapid turnover. We have used a chimeric gene composed mostly of growth hormone sequences with expression driven by the c-fos promoter to investigate the minimal sequence required to act as a functional destabilizing element and to monitor the effect of these sequences on early steps in the degradation pathway. We find that neither AUUUA, UAUUUA, nor AUUUAU can function as a destabilizing element. However, the sequence UAUUUAU, when present in three copies, is sufficient to destabilize a chimeric mRNA. We propose that this sequence functions by virtue of being a sufficient portion of the larger sequence, UUAUUUA(U/A)(U/A), that we propose forms the optimal binding site for a destabilizing factor. The destabilizing effect depends on the number of copies of this proposed binding site and their degree of mismatch in the first two and last two positions, with mismatches in the AUUUA sequence not being tolerated. We found a strict correlation between the effect of an ARE on degradation rate and the effect on the rate of poly(A) shortening, consistent with deadenylation being the first and rate-limiting step in degradation, and the step stimulated by destabilizing AREs. Deadenylation was observed to occur in at least two phases, with an oligo(A) intermediate transiently accumulating, consistent with the suggestion that the degradation processes may be similar in yeast and mammalian cells. AREs that are especially U rich and contain no UUAUUUA(U/A)(U/A) motifs failed to influence the degradation rate or the deadenylation rate, either when downstream of suboptimal destabilizing AREs or when alone.

Specificity and mechanism of thyroid hormone induction from an octamer response element.

Thyroid hormone response elements are specific DNA sequences that allow thyroid hormone receptors to confer ligand-dependent regulation of gene expression. The response elements characterized to date have been composed of varying arrangements of multiple copies of a conserved hexameric sequence. The traditional consensus half-site of these response elements is the sequence 5'-AGGTCA, although we have demonstrated recently that the optimal thyroid hormone receptor monomer binding site is 2 base pairs larger, 5'-TAAGGTCA. Since other members of this family of nuclear receptors also have been shown to use varying arrangements of the traditional hexamer sequence as response elements, we examined whether the octamer sequence was specific as a thyroid hormone response element. The studies reported here demonstrate that only thyroid hormone receptors confer ligand responsiveness to a reporter gene containing a single copy of the octamer sequence as a response element and that qualitative and quantitative differences in the binding of related nuclear receptors to this sequence can account for this functional specificity. We also have shown that thyroid hormone induction from the octamer response element occurs independently of retinoid X receptors, in contrast to the induction from traditional complex thyroid hormone response elements.

A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.

The recently cloned Clox (Cut-like homeo box) and CDP (CCAAT displacement protein), two mammalian counterparts of the Drosophila Cut homeo protein, correspond to alternatively spliced products of the same gene (mClox, for mammalian Cut-like homeo box). Although these proteins reportedly bind to apparently unrelated DNA sequences, we show by in vitro selection of optimal binding sites that both Clox and CDP have the same preferred DNA-binding specificity. The palindromic consensus target sequence, 5'-(t/a)(a/t)tATCGATTAt(t/c)(t/g)(t/a)-3', contains a bona fide homeo domain binding motif (ATTA). In addition, 37% of the in vitro-selected sequences have a CCAAT box, the canonical target for members of the family of CCAAT-binding factors. A characteristic feature of the cut homeo proteins is the presence of three evolutionarily conserved 73-amino-acid repeats of unknown function, the so-called cut repeats. We present evidence that the cut repeat II binds to mClox consensus targets independently of the DNA-binding activity of the homeo domain. In vitro selection of binding sites shows that the optimal targets for the cut repeat II contain one or more CCAAT boxes and, like the homeo domain, an ATTA core. These results indicate that the DNA-binding activity of the second cut repeat can account for the suggested role of CDP mClox as CCAAT displacement protein, a putative repressor of gene expression. We also report that the mClox homeo domain and cut repeat II interact in vitro in the absence of DNA. This interaction, which greatly enhances the DNA-binding activity of the binary complex, is specific to the cut homeo proteins. No cooperativity was observed between the cut repeat II and the homeo domains of Oct-1 and Gtx. Furthermore, the Drosophila cut repeat II, which does not appear to bind to DNA, also enhances the DNA-binding activity of the mClox homeo domain. Thus, the bifunctional cut repeat II, which defines a new family of bipartite DNA-binding proteins, is likely to play an important role in the function of the cut homeo proteins.

The RNA polymerase I transcription factor UBF is a sequence-tolerant HMG-box protein that can recognize structured nucleic acids.

Upstream Binding Factor (UBF) is important for activation of ribosomal RNA transcription and belongs to a family of proteins containing nucleic acid binding domains, termed HMG-boxes, with similarity to High Mobility Group (HMG) chromosomal proteins. Proteins in this family can be sequence-specific or highly sequence-tolerant binding proteins. We show that Xenopus UBF can be classified among the sequence-tolerant class. Methylation interference assays using enhancer DNA probes failed to reveal any critical nucleotides required for UBF binding. Selection by UBF of optimal binding sites among a population of enhancer oligonucleotides with randomized sequences also failed to reveal any consensus sequence. The minor groove specific drugs chromomycin A3, distamycin A and actinomycin D competed against UBF for enhancer binding, suggesting that UBF, like other HMG-box proteins, probably interacts with the minor groove. UBF also shares with other HMG box proteins the ability to bind synthetic cruciform DNA. However, UBF appears different from other HMG-box proteins in that it can bind both RNA (tRNA) and DNA. The sequence-tolerant nature of UBF-nucleic acid interactions may accommodate the rapid evolution of ribosomal RNA gene sequences.