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PubMed1995

Muscle gene E-box control elements. Evidence for quantitatively different transcriptional activities and the binding of distinct regulatory factors.

The muscle creatine kinase gene enhancer contains two regulatory elements (MCK-R and MCK-L) with the consensus E-box sequence (CAnnTG). A myocyte specific protein complex, MEF1, binds the MCK-R site. MEF1 contains several basic H-L-H myogenic determination factors (MDFs), each dimerized with ubiquitous members of the bH-L-H family (e.g. E12/E47). We now demonstrate that the ubiquitous bH-L-H factor E2-2 is a major component of the endogenous MCK-R site specific complex. Previous studies described the MCK-L site as a similar but low affinity MDF/bH-L-H heterodimer binding site. However, we find that the MCK-L site exhibits preferential binding of an unknown ubiquitous factor which contains neither E12/E47 nor E2-2, and that it exhibits differential transcriptional activity with muscle and non-muscle cells. The differential behavior of the MCK-L and MCK-R sites may be a general trait of E-box elements since one among several E-boxes in the MLC 1/3 enhancer also binds preferentially to the MCK-L factor. From our studies we now propose separate consensus sequences for MCK-R and MCK-L E-box types: AACAc/gc/gTGCa/t and GGa/cCANGTGGc/gNa/g. Our results suggest that while many muscle gene E-boxes are capable of binding the previously characterized spectrum of MDF/bH-L-H heterodimers in vitro, MCK-L type E-boxes probably bind qualitatively different factors in vivo.

Amino Acid Sequence↗

PubMed1996

Retinoid X receptor alters the determination of DNA binding specificity by the P-box amino acids of the thyroid hormone receptor.

Nuclear hormone receptors bind to hormone response elements in DNA consisting of two half-sites of 6 base pairs. The P-box amino acids of each receptor determine the identities of the central nucleotides of the half-site. 57 P-box variants of the human thyroid hormone receptor (hT3Rbeta) were used to demonstrate the relationship between P-box sequence and DNA binding specificity by homodimers and heterodimers formed with the retinoid X receptor (RXR). In general, the formation of heterodimers relieved many of the constraints on the compatibility of hT3Rbeta P-box sequences with DNA binding. Effects were most dramatic for heterodimers bound to a direct repeat spaced by four base pairs. RXR also overrides the P-box-derived DNA binding specificity of hT3Rbeta when heterodimers are bound to inverted or everted repeat elements. These effects of RXR are most pronounced on AGGTCA half-sites but are squelched when the RXR partner of the heterodimer is bound to an AGGACA half-site. The influence of RXR on hT3Rbeta DNA binding specificity varies with the orientation of half-sites in the element, the identity of the fourth base pair of the half-site, and the spacing between the half-sites of direct repeats. These differences suggest that the DNA binding domains of RXR-hT3Rbeta heterodimers are not positioned equivalently on the various elements, affecting the manner in which the P-box amino acids of hT3Rbeta interact with base pairs within the half-site.

Amino Acid Sequence↗

PubMed1996

Stimulation of mouse osteopontin promoter by v-Src is mediated by a CCAAT box-binding factor.

Osteopontin is an arginine-glycine-aspartic acid-containing cell adhesion protein, which is frequently expressed in transformed cells and is thought to play a role in tumorigenesis. v-Src is a transforming viral oncogene product encoded by Rous sarcoma virus (RSV). We report that v-Src expression in HT1080 fibrosarcoma cells significantly stimulates mouse osteopontin promoter activity. We also determined the v-Src response element in the osteopontin promoter as an inverted CCAAT box located at -53 to -49 from the transcription start site. Mutations of the CCAAT box disrupts protein-DNA interaction and diminishes both v-Src stimulation and basal promoter activity. A CCAAT box-containing fragment corresponding to -155 to -122 of RSV long terminal repeat competed with the -72 to -38 fragment of mouse osteopontin promoter for specific protein binding in the gel shift assay. A polyclonal antibody against CBF, a CCAAT box-binding factor, supershifted in gel shift assays the protein-DNA complex formed by nuclear extract of HT1080 with either the RSV CCAAT box fragment or with the osteopontin -72 to -38 fragment. Moreover, both osteopontin mRNA levels and enhancer activity of CCAAT box-containing -72 to -38 fragment were significantly elevated in v-src-transformed NIH 3T3 cells relative to parental cells. These findings suggest that the elevated osteopontin expression in transformed cells could be due, at least in part, to v-Src stimulation of the osteopontin promoter and that this effect is mediated by a CBF-like factor.

Animals↗

PubMed1996

Role of E box and initiator region in the expression of the rat follicle-stimulating hormone receptor.

The promoter for the rat follicle-stimulating hormone receptor (FSHR) gene contains a conserved consensus E box sequence and an initiator-like region (InR) sequence. Deletion analysis and transient transfections showed that a 114-base pair region (-143 to -30) that encompasses the E box and the InR was sufficient for greater than 75% of promoter function. DNase I footprint analysis showed that the E box and InR were protected by nuclear proteins from rat Sertoli cells, and the E box region was shown by electrophoretic mobility shift assays (EMSA) to be a site of Sertoli protein interactions. Mutations in the E box disrupted these interactions and reduced FSHR promoter activity. Co-transfection of the inhibitor of DNA binding (Id) with an FSHR/luciferase construct into mouse Sertoli 1 cells reduced FSHR promoter activity. Using EMSA, the upstream stimulatory factor was shown to be a component of the complexes that interacted with the E box in the FSHR promoter. Binding of proteins from rat Sertoli cells to the InR was demonstrated using EMSA. Also, an oligonucleotide that represented the sequence of the terminal deoxynucleotidyltransferase InR displaced the complexes at the FSHR InR. Mutations in the InR resulted in a significant reduction of FSHR promoter activity.

Animals↗

PubMed1998

GC- and E-box motifs as regulatory elements in the proximal promoter region of the neuronal nicotinic receptor alpha7 subunit gene.

The alpha7 subunit is a component of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors expressed in bovine adrenomedullary chromaffin cells. The proximal promoter of the gene coding for this subunit contains several GC-boxes and one E-box. Deletion analysis and transient transfections showed that a 120-base pair region (-77 to +43) including all of these elements gave rise to approximately 70 and 95% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of the different elements indicated that both GC and E motifs contribute to the activity of the alpha7 gene in a very prominent way. Using electrophoretic mobility shift assays, the upstream stimulatory factor (USF) was shown to be a component of the complexes that interacted with the E-box when nuclear extracts from chromaffin and SHSY-5Y cells were used. Binding of the early growth response gene transcription factor (Egr-1) to three different GC-boxes was also demonstrated by shift assays and DNase I footprint analysis. Likewise, alpha7 promoter activity increased by up to 5-fold when alpha7 constructs and an Egr-1 expression vector were cotransfected into chromaffin cell cultures. Mutagenesis of individual GC-boxes had little effect on Egr-1 activation. By contrast, pairwise suppression of GC-boxes abolished activation, especially when the most promoter-proximal of the Egr-1 sites was removed. Taken together, these studies indicate that the alpha7 gene is likely to be a target for multiple signaling pathways, in which various regulatory elements are involved.

Animals↗

PubMed1998

A novel F box protein, NFB42, is highly enriched in neurons and induces growth arrest.

NFB42 (neural F Box 42 kDa) is a novel gene product that is highly enriched in the nervous system. Its predicted protein contains an F box, a motif recently shown to couple cell cycle regulation to the proteasome pathway (Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J. W., and Elledge, S. (1996) Cell 86, 263-274). NFB42 mRNA and protein are expressed in all major areas of the adult rat brain but are not detected in non-neural tissues. NFB42 protein is localized primarily to the cytoplasm of neurons and does not appear to be present in glia. The presence of an F box in NFB42 suggests that it may be involved in cell cycle regulation; however, its expression in postmitotic neurons indicates that it is not involved in regulating typical cell cycle events. In an initial attempt to characterize the function of this protein, NFB42 was transfected into N1E-115 neuroblastoma and Chinese hamster ovary cells. The expression of full-length NFB42, but not an F box deletion mutant, inhibits proliferation in both cell lines. Additional experiments demonstrate that NFB42 interacts with Skp1p, a component of the proteasome pathway, and deletion of the F box also inhibits this interaction. Overall, the expression pattern of NFB42, along with the presence of an F box domain and the ability to inhibit growth, suggests that it may play a role in maintaining neurons in a postmitotic state.

Amino Acid Sequence↗

PubMed1999

The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transcription in skeletal muscle.

Previous experiments showed that activity of the -800-base pair MLC2slow promoter was 75-fold higher in the innervated soleus (SOL) compared with the noninnervated SOL muscles. Using in vivo DNA injection of MLC2slow promoter-luciferase constructs, the aim of this project was to identify regulatory sites and potential transcription factors important for slow nerve-dependent gene expression. Three sites within the proximal promoter (myocyte enhancer factor-2 (MEF2), E-box, and CACC box) were individually mutated, and the effect on luciferase expression was determined. There was no change in luciferase expression in the SOL and extensor digitorum longus (EDL) muscles when the E-box was mutated. In contrast, the MEF2 mutation resulted in a 30-fold decrease in expression in the innervated SOL muscles (10.3 versus 0.36 normalized relative light units (RLUs)). Transactivation of the MLC2slow promoter by overexpressing MEF2 was only seen in the innervated SOL (676,340 versus 2,225,957 RLUs; p < 0.01) with no effect in noninnervated SOL or EDL muscles. These findings suggest that the active MLC2slow promoter is sensitive to MEF2 levels, but MEF2 levels alone do not determine nerve-dependent expression. Mutation of the CACC box resulted in a significant up-regulation in the EDL muscles (0.23 versus 4.08 normalized RLUs). With the CACC box mutated, overexpression of MEF2 was sufficient to transactivate the MLC2slow promoter in noninnervated SOL muscles (27,536 versus 1, 605,797 RLUs). Results from electrophoretic mobility shift and supershift assays confirm MEF2 protein binding to the MEF2 site and demonstrate specific binding to the CACC sequence. These results suggest a model for nerve-dependent regulation of the MLC2slow promoter in which derepression occurs through the CACC box followed by quantitative expression through enhanced MEF2 activation.

Animals↗

PubMed2000

Mitogen-induced expression of the fibroblast growth factor-binding protein is transcriptionally repressed through a non-canonical E-box element.

The fibroblast growth factor-binding protein (FGF-BP) stimulates FGF-2-mediated angiogenesis and is thought to play an important role in the progression of squamous cell, colon, and breast carcinomas. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induction of the FGF-BP gene occurs through transcriptional mechanisms involving Sp1, AP-1, and CCAATT/enhancer-binding protein sites in the proximal FGF-BP gene promoter. The level of TPA induction, however, is limited due to the presence of a repressor element that shows similarity to a non-canonical E-box (AACGTG). Mutation or deletion of the repressor element led to enhanced induction by TPA or epidermal growth factor in cervical squamous cell and breast carcinoma cell lines. Repression was dependent on the adjacent AP-1 site, without discernible alteration in the binding affinity or composition of AP-1. We investigated the following two possible mechanisms for E-box-mediated repression: 1) CpG methylation of the core of the E-box element, and 2) binding of a distinct protein complex to this site. Point mutation of the CpG methylation site in the E-box showed loss of repressor activity. Conversely, in vitro methylation of this site significantly reduced TPA induction. In vitro gel shift analysis revealed distinct and TPA-dependent binding of USF1 and USF2 to the repressor element that required nucleotides within the E-box. Furthermore, chromatin immunoprecipitation assay showed that USF, c-Myc, and Max proteins were associated with the FGF-BP promoter in vivo. Overall, these findings suggested that the balance between trans-activation by AP-1 and repression through the E-box is an important control mechanism for fine-tuning the angiogenic response to growth factor-activated pathways.

CCAAT-Enhancer-Binding Proteins↗

PubMed2001

DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli.

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.

Bacterial Proteins↗

PubMed2002

A new class of transcription initiation factors, intermediate between TATA box-binding proteins (TBPs) and TBP-like factors (TLFs), is present in the marine unicellular organism, the dinoflagellate Crypthecodinium cohnii.

Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellate Crypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His(77), Arg(94), Tyr(171), Thr(188)) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5'-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii.

Amino Acid Sequence↗

PubMed2006

Polo box domain of Plk3 functions as a centrosome localization signal, overexpression of which causes mitotic arrest, cytokinesis defects, and apoptosis.

Polo-like kinase 3 (Plk3), an immediate early response gene product, plays an important role in the regulation of mitosis, DNA damage checkpoint activation, and Golgi dynamics. Similar to other members of the Plk family, Plk3 has a conserved kinase domain at the N terminus and a Polo box domain consisting of two Polo boxes at the C terminus. In this study, we demonstrate that the Polo box domain of Plk3 is sufficient for subcellular localization of this kinase to the centrosomes, the spindle poles, and the midbody when ectopically expressed in HeLa and U2OS cells. Both Polo boxes are required for the subcellular localization. Overexpression of the Polo box domain, not the kinase domain, of Plk3 causes significant cell cycle arrest and cytokinesis defects, eventually leading to mitotic catastrophe/apoptosis. Interestingly, the Polo box domain of Plk3 is more potent in inhibiting cell proliferation and inducing apoptosis than that of Plk1, suggesting that this domain can provide an additional structural basis for discovery of new anticancer drugs given the current emphasis on Plk1 as a therapeutic target.

Antineoplastic Agents↗

PubMed2005

Design of laying nests in furnished cages: influence of nesting material, nest box position and seclusion.

(1) Preferences for three nesting materials and nest box positions were investigated simultaneously in two trials using a furnished cage: one with 18 individual laying hens and one with 18 groups of 5 hens. Following a habituation period in pre-test cages, every hen or group of hens was tested for 2 d: once without and once with plastic flaps at the entrance of the nest boxes. (2) Hens preferred peat and artificial turf to coated wire mesh for egg laying. (3) One nest box position was clearly preferred to both other nest boxes. The hens' choice of nest box position was influenced by the pre-test cage in which they had been habituated. (4) The presence of plastic flaps at the entrance of the nest boxes had no influence on the proportion of eggs laid on the different nesting materials or on the proportion of floor eggs. (5) Individual and group testing resulted in the same overall results despite the presence of a distinct group effect.

Animal Husbandry↗

PubMed2002

CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA.

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric alpha-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.

Autoantigens↗

PubMed2003

A common sequence motif determines the Cajal body-specific localization of box H/ACA scaRNAs.

Post-transcriptional synthesis of 2'-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5' and 3' hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

Base Sequence↗

PubMed2003

E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome.

Myogenic determination factors are basic helix-loop-helix proteins that govern specification and differentiation of muscle cells, and bind to the E-box consensus sequence CANNTG in promoter regions of muscle-specific genes. No E-box mutation has been reported to date. RAPSN encodes rapsyn, a 43 kDa postsynaptic peripheral membrane protein that clusters the nicotinic acetylcholine receptor at the motor endplate. Transcriptional regulation mechanisms of RAPSN have not been studied. We here report two novel E-box mutations in the RAPSN promoter region in eight congenital myasthenic syndrome patients. Patient 1 carries -27C-->G that changes an E-box at -27 to -22 from CAGCTG to GAGCTG. An allele harboring -27C-->G is not transcribed in patient's muscle. Patients 2-8 are of Oriental Jewish stock of Iraqi or Iranian origin with facial malformations, and harbor -38A-->G that changes another E-box at -40 to -35 from CAACTG to CAGCTG, which does not affect the consensus CANNTG sequence. Haplotype analysis shows that -38A-->G arises from a common founder. For each mutation, position +1 represents the major transcriptional start site that we determine to be 172 nucleotides upstream of the translational start site. Electrophoretic mobility shift assays reveal that -38A-->G gains, and -27C-->G looses, binding affinity for different components of nuclear extracts of C2C12 myotubes. Luciferase reporter assays show that both -38A-->G and -27C-->G attenuate reporter gene expression in C2C12 myotubes, and that -27C-->G additionally attenuates reporter gene expression in MyoD- or myogenin-transfected HEK cells. The -27C-->G mutation also markedly attenuates the enhancer activity of an E-box on an SV40 promoter. Impaired transcriptional activities of the RAPSN promoter region predict reduced rapsyn expression and endplate acetylcholine receptor deficiency.

Adolescent↗

PubMed2003

Antiquity and evolution of the MADS-box gene family controlling flower development in plants.

MADS-box genes in plants control various aspects of development and reproductive processes including flower formation. To obtain some insight into the roles of these genes in morphological evolution, we investigated the origin and diversification of floral MADS-box genes by conducting molecular evolutionary genetics analyses. Our results suggest that the most recent common ancestor of today's floral MADS-box genes evolved roughly 650 MYA, much earlier than the Cambrian explosion. They also suggest that the functional classes T (SVP), B (and Bs), C, F (AGL20 or TM3), A, and G (AGL6) of floral MADS-box genes diverged sequentially in this order from the class E gene lineage. The divergence between the class G and E genes apparently occurred around the time of the angiosperm/gymnosperm split. Furthermore, the ancestors of three classes of genes (class T genes, class B/Bs genes, and the common ancestor of the other classes of genes) might have existed at the time of the Cambrian explosion. We also conducted a phylogenetic analysis of MADS-domain sequences from various species of plants and animals and presented a hypothetical scenario of the evolution of MADS-box genes in plants and animals, taking into account paleontological information. Our study supports the idea that there are two main evolutionary lineages (type I and type II) of MADS-box genes in plants and animals.

Animals↗

PubMed1990

Saturation mutagenesis of the Drosophila tRNA(Arg) gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation.

Transcription of eukaryotic tRNA genes is dependent on the A- and B-Box internal control regions (ICRs) and the upstream transcription modulatory region. The B-Box ICR spans nucleotides 52 to 62 and directs the primary binding of transcription factor C as the first step in the formation of a transcription complex. The conservation of the sequence of the B-Box in all tRNA species reflects its importance in both the expression of the gene and the processing, structure and function of the gene product. In order to identify the nucleotides essential to the promoter function of the B-Box ICR, site-directed mutagenesis was used to generate all the possible single point mutations at positions 52 to 58, 61 and 62 of a Drosophila melanogaster tRNA(Arg) gene. The effect of these mutations on gene transcription was evaluated using in vitro transcription and template exclusion competition assays. Optimal activity was displayed by the wild type tDNA(Arg) B-Box sequence but several other sequences supported in vitro transcription at wild type levels. The majority of mutants, however, showed lower efficiency in the in vitro transcription assay. Of the single point mutations, those at positions 53, 55, and 56 had a critical effect on gene function in Drosophila and HeLa transcription extracts and transcription factor interaction most likely requires base contacts at these positions. Since the effect of several of the point mutations cannot be explained in terms of possible major or minor groove contributions the possibility is raised that local DNA geometry also is an important determinant in specifying B-Box function.

Animals↗

PubMed1992

DnaA dependent replication of plasmid R1 occurs in the presence of point mutations that disrupt the dnaA box of oriR.

We have found that DnaA dependent replication of R1 still occurred when 5 of the 9 bases in the dnaA box present in oriR were changed by site directed mutagenesis although the replication efficiency decreased to 20% and 70% of the wild-type origin in vitro and in vivo respectively. Additional mutation of a second dnaA box, 28 bp upstream oriR, that differs in only one base from the consensus sequence, did not affect the level of replication whereas polyclonal antibodies against DnaA totally abolished in vitro replication in the absence of the dnaA box. Wild-type RepA as well as a RepA mutant, RepA2623, that binds to oriR but that is inactive in promoting in vitro replication of plasmid R1, induce efficient binding of DnaA to the dnaA box. However, specific binding of DnaA to oriR was not detected by DNase I protection experiments in the absence of the dnaA box. These results suggest that the entrance of the DnaA protein in oriR is promoted initially by interactions with a RepA-oriR pre-initiation complex and that, in the absence of the dnaA box, these interactions can support, with reduced efficiency, DnaA dependent replication of plasmid R1.

Bacterial Proteins↗