SMART MODE:

NORMAL GENOMIC

• Morotomi-Yano K, Yano K, Saito H, Sun Z, Iwama A, Miki Y
• Human regulatory factor X 4 (RFX4) is a testis-specific dimeric DNA-binding protein that cooperates with other human RFX members.
• J Biol Chem. 2002; 277: 836-42
• Display abstract

• Regulatory factor X (RFX) members are evolutionarily conserved transcription factors that share a highly conserved winged helix DNA-binding domain. Human RFX4 has been isolated from breast cancer as a partial cDNA encoding a short RFX-type DNA-binding domain fused to the estrogen receptor, but the entire structure of RFX4 has been unknown. Here, we report the molecular cloning and characterization of human RFX4. RFX4 contains evolutionarily conserved regions, including a RFX-type DNA-binding domain, a dimerization domain, and other conserved regions, and is closely related to RFX1, RFX2, and RFX3 in structure. The expression of RFX4 is restricted to testis. In vitro synthesized RFX4 protein bound to typical RFX binding sites in a sequence-dependent manner. Immunoprecipitation analyses showed that RFX4 interacts physically with RFX2, RFX3, and RFX4 itself but not with RFX1. In contrast to other mammalian RFX members that form dimers, RFX4 is revealed to have no distinct transcriptional activation domains. By using a chimeric protein of RFX1 and RFX4, the C-terminal domain of RFX4 was shown to be a possible transcriptional repression domain. Taken together, these results indicate that RFX4 is the first mammalian member of RFX family without transcriptional activation capacity and might function through selective interactions with other RFX members in transcriptional regulation.

• Loguercio Polosa P, Megli F, Di Ponzio B, Gadaleta MN, Cantatore P, Roberti M
• Cloning of two sea urchin DNA-binding proteins involved in mitochondrial DNA replication and transcription.
• Gene. 2002; 286: 113-20
• Display abstract

• The cloning of the cDNA for two mitochondrial proteins involved in sea urchin mtDNA replication and transcription is reported here. The cDNA for the mitochondrial D-loop binding protein (mtDBP) from the sea urchin Strongylocentrotus purpuratus has been cloned by a polymerase chain reaction-based approach. The protein displays a very high similarity with the Paracentrotus lividus homologue as it contains also the two leucine zipper-like domains which are thought to be involved in intramolecular interactions needed to expose the two DNA binding domains in the correct position for contacting DNA. The cDNA for the mitochondrial single-stranded DNA-binding protein (mtSSB) from P. lividus has been also cloned by a similar approach. The precursor protein is 146 amino acids long with a presequence of 16 residues. The deduced amino acid sequence shows the highest homology with the Xenopus laevis protein and the lowest with the Drosophila mtSSB. The computer modeling of the tertiary structure of P. lividus mtSSB shows a structure very similar to that experimentally determined for human mtSSB, with the conservation of the main residues involved in protein tetramerization and in DNA binding.

• Schubot FD, Chen CJ, Rose JP, Dailey TA, Dailey HA, Wang BC
• Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription.
• Protein Sci. 2001; 10: 1980-8
• Display abstract

• Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined. In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution. The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices. Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC'. This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter. Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site.

• Prieto-Martin A, Montoya J, Martinez-Azorin F
• A study on the human mitochondrial RNA polymerase activity points to existence of a transcription factor B-like protein.
• FEBS Lett. 2001; 503: 51-5
• Display abstract

• In the present work, the RNA polymerase activity of the human mitochondrial RNA polymerase mature protein (h-mtRPOLm) is shown, and its molecular activity calculated (2.1+/-0.9 min(-1)). An activity analysis of h-mtRPOLm and deleted versions of it has demonstrated that the entire recombinant protein is required for this activity. In addition, h-mtRPOLm alone or in presence of the known mitochondrial transcription factors (human mitochondrial transcription factor A and/or human mitochondrial transcription termination factor) is not able to initiate transcription from the specific human mitochondrial promoters pointing to the existence of a human mitochondrial transcription factor B-like protein.

• Hollenbeck JJ, Gurnon DG, Fazio GC, Carlson JJ, Oakley MG
• A GCN4 variant with a C-terminal basic region binds to DNA with wild-type affinity.
• Biochemistry. 2001; 40: 13833-9
• Display abstract

• Basic-region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a leucine zipper dimerization domain and a basic region that directly contacts DNA. In all naturally occurring bZip proteins, the basic region is positioned N-terminal to the leucine zipper. We have designed a series of model bZip peptides in which the basic region of the yeast transcriptional activator GCN4 is placed C-terminal to its leucine zipper. DNA-binding studies demonstrate that the optimal reverse GCN4 (rGCN4) peptide is able to bind specifically and with wild-type affinity to DNA despite this unnatural arrangement of the two subdomains. These results suggest that a thermodynamic basis for the observed N-terminal positioning of the basic region relative to the dimerization domain is unlikely.

• Johannesson H, Wang Y, Engstrom P
• DNA-binding and dimerization preferences of Arabidopsis homeodomain-leucine zipper transcription factors in vitro.
• Plant Mol Biol. 2001; 45: 63-73
• Display abstract

• Homeodomain-leucine zipper (HDZip) proteins constitute a large family of transcription factors apparently unique to plants. In this report we characterize the DNA-binding and dimerization preferences in vitro of class I HDZip proteins. Using gel-exclusion chromatography and in vitro protein binding assays we demonstrate that the HDZip class I protein ATHB5 forms a homodimeric complex in solution. Consistent with this finding we have demonstrated the sequence-specific interaction of ATHB5 with a 9 bp pseudopalindromic DNA sequence, CAATNATTG, composed of two half-sites overlapping at a central position, by use of a PCR-assisted binding-site selection assay and competitive EMSA experiments. A majority of other known members of HDZip class I interacted with similar DNA sequences, but differed in their preference for A/T versus G/C in the central position of the binding site. Selective heterodimerization in vitro was demonstrated between ATHB5 and different class I HDZip proteins. Heterodimer formation between class I HDZip proteins is of potential functional significance for the integration of information from different signalling pathways in the control of plant development.

• Rodeheffer MS, Boone BE, Bryan AC, Shadel GS
• Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase.
• J Biol Chem. 2001; 276: 8616-22
• Display abstract

• Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.

• Maxon ME, Herskowitz I
• Ash1p is a site-specific DNA-binding protein that actively represses transcription.
• Proc Natl Acad Sci U S A. 2001; 98: 1495-500
• Display abstract

• ASH1 encodes a protein that is localized specifically to the daughter cell nucleus, where it has been proposed to repress transcription of the HO gene. Using Ash1p purified from baculovirus-infected insect cells, we have shown that Ash1p binds specific DNA sequences in the HO promoter. DNase I protection analyses showed that Ash1p recognizes a consensus sequence, YTGAT. Mutation of this consensus abolishes Ash1p DNA binding in vitro. We have shown that Ash1p requires an intact zinc-binding domain in its C terminus for repression of HO in vivo and that this domain may be involved in DNA binding. A heterologous DNA-binding domain fused to an N-terminal segment of Ash1p functions as an active repressor of transcription. Our studies indicate that Ash1p is a DNA-binding protein of the GATA family with a separable transcriptional repression domain.

• Wang Y, Devereux W, Stewart TM, Casero RA Jr
• Characterization of the interaction between the transcription factors human polyamine modulated factor (PMF-1) and NF-E2-related factor 2 (Nrf-2) in the transcriptional regulation of the spermidine/spermine N1-acetyltransferase (SSAT) gene.
• Biochem J. 2001; 355: 45-9
• Display abstract

• Polyamines and polyamine analogues have been demonstrated to modulate the transcription of various genes. Spermidine/spermine N(1)-acetyltransferase (SSAT) is transcriptionally regulated through the interaction of at least two trans-acting transcription factors, NF-E2-related factor 2 (Nrf-2) and PMF-1 (polyamine modulated factor-1). Nrf-2 has previously been shown to regulate transcription of other genes through interactions between its C-terminal leucine zipper and the leucine-zipper region of other members of the small Maf protein family (the term "Maf" is derived from MusculoAponeurotic-Fibrosarcoma virus). Here it is demonstrated that the interaction between Nrf-2 and PMF-1 is mediated through the binding of the leucine-zipper region of Nrf-2 and a C-terminal coiled-coil region of PMF-1 that does not contain a leucine zipper. Mutations that interrupt either the leucine zipper of Nrf-2 or the coiled-coil region of PMF-1 are demonstrated to alter the ability of these factors to interact, thus their ability to regulate the transcription of the SSAT gene is lost.

• Selwood SP, Chrzanowska-Lightowlers ZM, Lightowlers RN
• Does the mitochondrial transcription-termination complex play an essential role in controlling differential transcription of the mitochondrial DNA?
• Biochem Soc Trans. 2000; 28: 154-9
• Display abstract

• The mechanism of mitochondrial transcription is well documented although the method of regulation remains obscure. The mitochondrial transcription-termination complex, mTERF, holds a key position in determining the fate of heavy-strand promotor-initiated transcripts and has been suggested as a candidate in the regulation of mitochondrial DNA (mtDNA) transcription. We report here the first example of a modulation of mTERF-complex binding activity concomitant with a differential mtDNA transcription rate. We suggest that these observations are indicative of a method of intra-organellar transcriptional fine tuning.

• Schmitt EK, Kuck U
• The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors.
• J Biol Chem. 2000; 275: 9348-57
• Display abstract

• Here we report the isolation and characterization of a novel transcription factor from the cephalosporin C-producing fungus Acremonium chrysogenum. We have identified a protein binding site in the promoter of the beta-lactam biosynthesis gene pcbC, located 418 nucleotides upstream of the translational start. Using the yeast one-hybrid system, we succeeded in isolating a cDNA clone encoding a polypeptide, which binds specifically to the pcbC promoter. The polypeptid shows significant sequence homology to human transcription factors of the regulatory factor X (RFX) family and was designated CPCR1. A high degree of CPCR1 binding specificity was observed in in vivo and in vitro experiments using mutated versions of the DNA binding site. The A. chrysogenum RFX protein CPCR1 recognizes an imperfect palindrome, which resembles binding sites of human RFX transcription factors. One- and two-hybrid experiments with truncated versions of CPCR1 showed that the protein forms a DNA binding homodimer. Nondenaturing electrophoresis revealed that the CPCR1 protein exists in vitro solely in a multimeric, probably dimeric, state. Finally, we isolated a homologue of the cpcR1 gene from the penicillin-producing fungus Penicillium chrysogenum and determined about 60% identical amino acid residues in the DNA binding domain of both fungal RFX proteins, which show an overall amino acid sequence identity of 29%.

• Fujii Y, Shimizu T, Toda T, Yanagida M, Hakoshima T
• Structural basis for the diversity of DNA recognition by bZIP transcription factors.
• Nat Struct Biol. 2000; 7: 889-93
• Display abstract

• The basic region leucine zipper (bZIP) proteins form one of the largest families of transcription factors in eukaryotic cells. Despite relatively high homology between the amino acid sequences of the bZIP motifs, these proteins recognize diverse DNA sequences. Here we report the 2.0 A resolution crystal structure of the bZIP motif of one such transcription factor, PAP1, a fission yeast AP-1-like transcription factor that binds DNA containing the novel consensus sequence TTACGTAA. The structure reveals how the Pap1-specific residues of the bZIP basic region recognize the target sequence and shows that the side chain of the invariant Asn in the bZIP motif adopts an alternative conformation in Pap1. This conformation, which is stabilized by a Pap1-specific residue and its associated water molecule, recognizes a different base in the target sequence from that in other bZIP subfamilies.

• Musicco C et al.
• Regulation of the expression of the sea urchin mitochondrial D-loop binding protein during early development.
• Biochem Biophys Res Commun. 2000; 277: 299-304
• Display abstract

• The Paracentrotus lividus mitochondrial D-loop binding protein (mtDBP) is a DNA-binding protein which is involved in the regulation of sea urchin mtDNA transcription. Immunoblots of Heparin Sepharose-bound proteins at selected early developmental stages, as well as electrophoretic mobility shift assay, show that mtDBP is present in the egg at a concentration of about 1 x 10(6) molecules/egg. Its level increases after fertilization of about twofold, remaining substantially unchanged between 16-h blastula stage and early pluteus stage and declines thereafter. The content of mtDBP mRNA, determined by RNase protection experiments, increases about sevenfold at the 16-h blastula stage compared to the egg. A considerable decrease occurs at the 40-h pluteus stage, which precedes that of the protein. These results suggest that the expression of mtDBP is regulated at transcriptional level up to blastula stage, while other factors, in addition to the level of the RNA, may control the content of this protein in the following stages of embryogenesis.

• Hollenbeck JJ, Oakley MG
• GCN4 binds with high affinity to DNA sequences containing a single consensus half-site.
• Biochemistry. 2000; 39: 6380-9
• Display abstract

• bZip proteins contain a bipartite DNA-binding motif consisting of a "leucine zipper" dimerization domain and a highly charged "basic region" that directly contacts DNA. These transcription factors form dimeric complexes with each monomer recognizing half of a symmetric or nearly symmetric DNA site. We have found that the bZip protein GCN4 can also bind with high affinity to DNA sites containing only a single GCN4 consensus half-site. Because several recent lines of evidence have suggested a role for monomeric DNA binding by bZip proteins, we investigated the structure of the GCN4.half-site complex. Quantitative DNA binding and affinity cleaving studies support a model in which GCN4 binds as a dimer, with one monomer making specific contacts to the consensus half-site and the other monomer forming nonspecific contacts that are nonetheless important for binding affinity. We also examined the folding transition induced in the basic regions of this complex upon binding DNA. Circular dichroism (CD) studies demonstrate that the basic regions of both monomers are helical, suggesting that a protein folding transition may be required for both specific and nonspecific DNA binding by GCN4.

• Cliften PF, Jang SH, Jaehning JA
• Identifying a core RNA polymerase surface critical for interactions with a sigma-like specificity factor.
• Mol Cell Biol. 2000; 20: 7013-23
• Display abstract

• Cyclic interactions occurring between a core RNA polymerase (RNAP) and its initiation factors are critical for transcription initiation, but little is known about subunit interaction. In this work we have identified regions of the single-subunit yeast mitochondrial RNAP (Rpo41p) important for interaction with its sigma-like specificity factor (Mtf1p). Previously we found that the whole folded structure of both polypeptides as well as specific amino acids in at least three regions of Mtf1p are required for interaction. In this work we started with an interaction-defective point mutant in Mtf1p (V135A) and used a two-hybrid selection to isolate suppressing mutations in the core polymerase. We identified suppressors in three separate regions of the RNAP which, when modeled on the structure of the closely related phage T7 RNAP, appear to lie on one surface of the protein. Additional point mutations and biochemical assays were used to confirm the importance of each region for Rpo41p-Mtf1p interactions. Remarkably, two of the three suppressors are found in regions required by T7 RNAP for DNA sequence recognition and promoter melting. Although these essential regions of the phage RNAP are poorly conserved with the mitochondrial RNAPs, they are conserved among the mitochondrial enzymes. The organellar RNAPs appear to use this surface in an alternative way for interactions with their separate sigma-like specificity factor, which, like its bacterial counterpart, provides promoter recognition and DNA melting functions to the holoenzyme.

• Gurezka R, Laage R, Brosig B, Langosch D
• A heptad motif of leucine residues found in membrane proteins can drive self-assembly of artificial transmembrane segments.
• J Biol Chem. 1999; 274: 9265-70
• Display abstract

• Specific interactions between alpha-helical transmembrane segments are important for folding and/or oligomerization of membrane proteins. Previously, we have shown that most transmembrane helix-helix interfaces of a set of crystallized membrane proteins are structurally equivalent to soluble leucine zipper interaction domains. To establish a simplified model of these membrane-spanning leucine zippers, we studied the homophilic interactions of artificial transmembrane segments using different experimental approaches. Importantly, an oligoleucine, but not an oligoalanine, se- quence efficiently self-assembled in membranes as well as in detergent solution. Self-assembly was maintained when a leucine zipper type of heptad motif consisting of leucine residues was grafted onto an alanine host sequence. Analysis of point mutants or of a random sequence confirmed that the heptad motif of leucines mediates self-recognition of our artificial transmembrane segments. Further, a data base search identified degenerate versions of this leucine motif within transmembrane segments of a variety of functionally different proteins. For several of these natural transmembrane segments, self-interaction was experimentally verified. These results support various lines of previously reported evidence where these transmembrane segments were implicated in the oligomeric assembly of the corresponding proteins.

• Hurlin PJ, Steingrimsson E, Copeland NG, Jenkins NA, Eisenman RN
• Mga, a dual-specificity transcription factor that interacts with Max and contains a T-domain DNA-binding motif.
• EMBO J. 1999; 18: 7019-28
• Display abstract

• The basic-helix-loop-helix-leucine zipper (bHLHZip) proteins Myc, Mad and Mnt are part of a transcription activation/repression system involved in the regulation of cell proliferation. The function of these proteins as transcription factors is mediated by heterodimerization with the small bHLHZip protein Max, which is required for their specific DNA binding to E-box sequences. We have identified a novel Max-interacting protein, Mga, which contains a Myc-like bHLHZip motif, but otherwise shows no relationship with Myc or other Max-interacting proteins. Like Myc, Mad and Mnt proteins, Mga requires heterodimerization with Max for binding to the preferred Myc-Max-binding site CACGTG. In addition to the bHLHZip domain, Mga contains a second DNA-binding domain: the T-box or T-domain. The T-domain is a highly conserved DNA-binding motif originally defined in Brachyury and characteristic of the Tbx family of transcription factors. Mga binds the preferred Brachyury-binding sequence and represses transcription of reporter genes containing promoter-proximal Brachyury-binding sites. Surprisingly, Mga is converted to a transcription activator of both Myc-Max and Brachyury site-containing reporters in a Max-dependent manner. Our results suggest that Mga functions as a dual-specificity transcription factor that regulates the expression of both Max-network and T-box family target genes.

• Ikeda TM, Gray MW
• Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria.
• Mol Cell Biol. 1999; 19: 8113-22
• Display abstract

• To investigate the transcriptional apparatus in wheat mitochondria, mitochondrial extracts were subjected to column chromatography and protein fractions were analyzed by in vitro transcription and mobility shift assays. Fractions eluting from DEAE-Sephacel between 0.2 and 0.3 M KCl displayed DNA-binding activity and supported specific transcription initiated from a wheat cox2 promoter. The active DEAE-Sephacel pool was further resolved by chromatography on phosphocellulose. Fractions that exhibited DNA-binding activity and that stimulated both specific and nonspecific transcription in vitro were highly enriched in a 63-kDa protein (p63). From peptide sequence obtained from purified p63, a cDNA encoding the protein was assembled. The predicted amino acid sequence (612 amino acid residues, 69 kDa) contains a basic N-terminal targeting sequence expected to direct transport of the protein into mitochondria. The p63 sequence also features an acidic domain characteristic of transcriptional activation factors, as well as sequence blocks displaying limited similarity to positionally equivalent regions in sigma factors from eubacteria related to mitochondria. Recombinant p63 possesses DNA-binding activity, exhibiting an affinity for the core cox2 promoter element and upstream regions in gel shift assays and having the ability to enhance specific transcription in vitro. Transcripts encoding p63 are expressed at an early stage in the germination of isolated wheat embryos, in a temporal pattern parallelling that of newly synthesized precursors of cox2, a mitochondrial gene. Taken together, these data suggest a role for p63 in transcription in wheat mitochondria.

• Roberti M et al.
• In vivo mitochondrial DNA-protein interactions in sea urchin eggs and embryos.
• Curr Genet. 1999; 34: 449-58
• Display abstract

• Footprinting studies with the purine-modifying agent dimethyl sulphate were performed in Paracentrotus lividus eggs and embryos to analyze in vivo the interactions between protein and mitochondrial DNA. Footprinting in the small non-coding region and at the boundary between the ND5 and ND6 genes revealed two strong contact sites corresponding with the in vitro binding sequences of mitochondrial D-loop-Binding Protein (mtDBP). The analysis of the pause region of mtDNA replication showed a strong footprint corresponding with the binding site of the mitochondrial Pause region-Binding Protein-2 (mtPBP-2), but only a very weak signal at the binding site of the mitochondrial Pause region-Binding Protein-1 (mtPBP-1), which in vitro binds DNA with high efficiency. In vitro and in vivo analysis of the 3' end-region of the two rRNA genes showed no significant protein-DNA interactions, suggesting that, in contrast to mammals, the 3' ends of sea urchin mitochondrial rRNAs are not generated by a protein-dependent transcription termination event. These and other data support a model in which expression of mitochondrial genes in sea urchins is regulated post-transcriptionally. Footprinting at the five AT-rich consensus regions allowed the detection of a binding site in the non-coding region for an as-yet unidentified protein, mtAT-1BP. The occupancy of this site appears to be developmentally regulated, being detectable in the pluteus larval stage, but not in unfertilized eggs.

• Kohler JJ, Metallo SJ, Schneider TL, Schepartz A
• DNA specificity enhanced by sequential binding of protein monomers.
• Proc Natl Acad Sci U S A. 1999; 96: 11735-9
• Display abstract

• Transcriptional activation often requires the rapid assembly of complexes between dimeric transcription factors and specific DNA sites. Here we show that members of the basic region leucine zipper and basic region helix-loop-helix zipper transcription factor families follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA. Nonspecific protein or DNA competitors have little effect on the rate of assembly along this pathway, but slow a competing pathway in which preformed dimers bind DNA. The sequential monomer-binding pathway allows the protein to search for and locate a specific DNA site more quickly, resulting in greater specificity prior to equilibrium.

• Fujii Y, Shimizu T, Kusumoto M, Kyogoku Y, Taniguchi T, Hakoshima T
• Crystal structure of an IRF-DNA complex reveals novel DNA recognition and cooperative binding to a tandem repeat of core sequences.
• EMBO J. 1999; 18: 5028-41
• Display abstract

• There has been growing interest in the role of the IRF (interferon regulatory factor) family of transcription factors in the regulation of immune responses, cytokine signaling, and oncogenesis. These members are characterized by their well-conserved DNA binding domains at the N-terminal regions. Here we report the 2.2 A resolution crystal structure of the DNA binding domain of one such family member, IRF-2, bound to DNA. The structure reveals its recognition sequence, AANNGAAA (here, recognized bases are underlined and in bold, and N indicates any base), and its cooperative binding to a tandem repeat of the GAAA core sequence induced by DNA structure distortions. These facts explain well the diverse binding properties of the IRF family members, which bind to both single and tandemly repeated sequences. Furthermore, we also identified the 'helix-hairpin-strand motif' at the C terminus of the recognition helix as a metal binding site that is commonly found in certain classes of DNA-interactive proteins. Our results provide new insights into the structure and function of this family of transcription factors.

• Biswas EE, Biswas SB
• Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.
• Biochemistry. 1999; 38: 10929-39
• Display abstract

• We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.

• Virbasius CM, Wagner S, Green MR
• A human nuclear-localized chaperone that regulates dimerization, DNA binding, and transcriptional activity of bZIP proteins.
• Mol Cell. 1999; 4: 219-28
• Display abstract

• We have identified and cloned a human nuclear protein that dramatically increases DNA binding of transcription factors that contain a basic region-leucine zipper (bZIP) DNA binding domain. We show that this bZIP enhancing factor (BEF) functions as a molecular chaperone. BEF stimulates DNA binding by recognizing the unfolded leucine zipper and promoting the folding of bZIP monomers to dimers; the elevated concentration of the bZIP dimer then drives the DNA binding reaction. Antisense experiments indicate that BEF is required for efficient transcriptional activation by bZIP proteins in vivo. Our results reveal protein folding in the nucleus as a step at which sequence-specific DNA binding proteins can be regulated.

• Roberti M, Musicco C, Polosa PL, Milella F, Gadaleta MN, Cantatore P
• Multiple protein-binding sites in the TAS-region of human and rat mitochondrial DNA.
• Biochem Biophys Res Commun. 1998; 243: 36-40
• Display abstract

• To study the molecular mechanisms responsible for the regulation of mitochondrial DNA copy number, in vivo and in organello dimethyl sulfate footprinting experiments in human fibroblasts and rat liver mitochondria were carried out. By this approach we identified in both species two specific protein binding sites in the 3' region of the displacement loop of mitochondrial DNA. One site contains the TAS-D element of human and rat mitochondrial DNA; the other covers TAS-C and TAS-B in human, whereas in rat it comprises part of TAS-B. We suggest that the protected sequences might be the site of action of protein factors involved in the premature termination of mitochondrial DNA heavy-strand synthesis.

• Roberti M, Musicco C, Loguercio Polosa P, Gadaleta MN, Quagliariello E, Cantatore P
• Purification and characterization of a mitochondrial, single-stranded-DNA-binding protein from Paracentrotus lividus eggs.
• Eur J Biochem. 1997; 247: 52-8
• Display abstract

• A binding protein for single-stranded DNA was purified from Paracentrotus lividus egg mitochondria to near homogeneity by chromatography on DEAE-Sephacel and single-stranded-DNA-cellulose. The protein consists of a single polypeptide of about 15 kDa. Glycerol gradient sedimentation analysis suggested that P. lividus mitochondrial single-stranded-DNA-binding protein exists as a homo-oligomer, possibly a tetramer, in solution. The protein shows a stronger preference for poly(dT) with respect to single-stranded M13, poly(dI) and poly(dC). Binding to poly(dA) takes place with much lower affinity. The binding-site size, determined by gel mobility-shift experiments with oligonucleotides of different length, is approximately 45 nucleotides. The binding to single-stranded DNA occurs with low or no cooperativity and is not influenced by ionic strength. The protein has a very high affinity for the DNA: its apparent macroscopic association constant is 2x10(9) M(-1), a value which is the highest among the mitochondrial single-stranded-DNA-binding proteins characterized to date. The lack of cooperativity and the high association constant represent distinctive features of this protein and might be related to the peculiar mechanism of sea urchin mitochondrial DNA replication.

• Inagaki H, Matsushima Y, Ohshima M, Kitagawa Y
• Interferons suppress mitochondrial gene transcription by depleting mitochondrial transcription factor A (mtTFA).
• J Interferon Cytokine Res. 1997; 17: 263-9
• Display abstract

• Mitochondrial gene transcription activity in organello was suppressed after culturing HeLa cells with 1000 U/ml of interferon-alpha (IFN-alpha) or IFN-gamma for 18 h. The suppression was associated with reduced levels of mitochondrial gene transcripts. Northern blot analysis of HeLa cell RNA showed marked reduction of the mRNA encoding for mitochondrial transcription factor A (mtTFA). Immunoblotting with antiserum directed against recombinant mtTFA showed a reduced level of the protein as well. Consistently, gel-retardation assay of mitochondrial extract showed reduced level of functional mtTFA, which is known to bind to both heavy and light strand promoters of bidirectionally transcribed mitochondrial DNA. We suggest that depletion of mtTFA is a pathway through which IFNs depress the mitochondrial respiration.

• Micol V, Fernandez-Silva P, Attardi G
• Functional analysis of in vivo and in organello footprinting of HeLa cell mitochondrial DNA in relationship to ATP and ethidium bromide effects on transcription.
• J Biol Chem. 1997; 272: 18896-904
• Display abstract

• In vivo and in organello footprinting techniques based on methylation interference have been utilized to investigate protein-DNA interactions in the transcription initiation and rDNA transcription termination regions of human mitochondrial DNA (mtDNA) in functionally active mitochondria. In particular, the changes in methylation reactivity of these regions in response to treatment of the organelles with ATP or ethidium bromide, which affects differentially the rates of mitochondrial rRNA and mRNA synthesis, have been analyzed. Two major sites of protein-DNA interactions have been identified in the main control region of mtDNA, both in vivo and in organello, which correspond to the regions of the light-strand promoter and heavy-strand rRNA-specific promoter. The in organello footprinting of the latter showed ATP- and ethidium bromide-dependent modifications that could be correlated with changes in the rate of rRNA but not of mRNA synthesis. By contrast, no ATP effects were observed on the in organello footprinting pattern of the termination region and on in vitro transcription termination, strongly suggesting that ATP control of rRNA synthesis occurs at the initiation level. Several methylation interference sites were found upstream of the whole H-strand transcription unit, pointing to possible protein-DNA interactions related to the activity of this unit. In vivo footprinting of the rDNA transcription termination region of human mtDNA has revealed a very strong protection pattern, indicating a high degree of occupancy of the termination site by mitochondrial transcription termination factor (approximately 80%).

• Shao X, Tarnasky HA, Schalles U, Oko R, van der Hoorn FA
• Interactional cloning of the 84-kDa major outer dense fiber protein Odf84. Leucine zippers mediate associations of Odf84 and Odf27.
• J Biol Chem. 1997; 272: 6105-13
• Display abstract

• The study of mammalian sperm tail outer dense fibers (ODF), a structure of unknown function, is hampered by the insoluble nature of ODF proteins and the availability of only one cloned component, Odf27. We report here the first use of the Odf27 leucine zipper as bait in a yeast two-hybrid screen to isolate a novel testis-specific protein whose interaction with Odf27 depends critically on the Odf27 leucine zipper. We find that the novel gene, 111-450, encodes a product that localizes to ODF as determined by fluorescence microscopy and immunoelectron microscopy and that the gene 111-450 product is identical to the major ODF protein, Odf84. Interestingly, Odf84 contains two C-terminal leucine zippers, and we demonstrate that all leucine residues in the upstream leucine zipper are required for interaction with Odf27, demonstrating the strategic validity of our approach. The use of the yeast screening approach to isolate leucine zipper containing proteins should be useful in other systems, and our findings have implications for ODF structural models.

• Suzuki H, Suzuki S, Sakurai T, Kumar S, Ozawa T
• A bovine mtDNA-binding protein to a conserved sequence adjacent to the termination associated sequence in the vertebrate mitochondrial displacement loop region.
• Biochem Mol Biol Int. 1996; 38: 275-83
• Display abstract

• We have found a sequence-specific mitochondrial (mt) DNA-binding protein in bovine mitochondria. The binding site designated Mt5 is adjacent to the termination associated sequence in the vertebrate mt displacement loop region and is interspecifically conserved. An UV-crosslinking and gel filtration demonstrated that a Mt5-binding protein (designated Mt5BP) exists in the mitochondria as a homodimer of an approximately 97-kDa polypeptide. The nucleotide sequence of Mt5 is homologous to that of the binding site for the recently reported mt pause-region binding protein (mtPBP) 1 from sea urchin embryo mitochondria. Gel mobility shift competition experiments indicated that Mt5BP can recognize human sequence couterparts for the mtPBP1-binding site. These results suggest that Mt5BP is related to mtPBP1 with respect to discontinuance of heavy-strand synthesis.

• Hirst JD, Vieth M, Skolnick J, Brooks CL 3rd
• Predicting leucine zipper structures from sequence.
• Protein Eng. 1996; 9: 657-62
• Display abstract

• The leucine zipper structure is adopted by one family of the coiled coil proteins. Leucine zippers have a characteristic leucine repeat: Leu-X6-Leu-X6-Leu-X6-Liu (where X may be any residue). However, many sequences have the leucine repeat, but do not adopt the leucine zipper structure (we shall refer to these as non-zippers). We have found and analyzed residue pair patterns that allow one to identify correctly 90% of leucine zippers and 97% of non-zippers. Simpler analyses, based on the frequency of occurrence of residues at certain positions, specify, at most, 65% of zippers and 80-90% of non-zippers. Both short and long patterns contribute to the successful discrimination of leucine zippers from non-zippers. A number of these patterns involve hydrophobic residues that would be placed on the solvent-exposed surface of the helix, were the sequence to adopt a leucine zipper structure. Thus, an analysis of protein sequences has allowed us to improve discrimination between leucine zippers and non-zippers, and has provided some further insight into the physical factors influencing the leucine zipper structure.

• Micol V, Fernandez-Silva P, Attardi G
• Isolation and assay of mitochondrial transcription termination factor from human cells.
• Methods Enzymol. 1996; 264: 158-73

• Cantatore P, Daddabbo L, Fracasso F, Gadaleta MN
• Identification by in Organello footprinting of protein contact sites and of single-stranded DNA sequences in the regulatory region of rat mitochondrial DNA. Protein binding sites and single-stranded DNA regions in isolated rat liver mitochondria.
• J Biol Chem. 1995; 270: 25020-7
• Display abstract

• Footprinting studies with the purine-modifying reagent dimethyl sulfate and with the single-stranded DNA probing reagent potassium permanganate were carried out in isolated mitochondria from rat liver. Dimethyl sulfate footprinting allowed the detection of protein-DNA interactions within the rat analogues of the human binding sites for the transcription termination factor mTERF and for the transcription activating factor mt-TFA. Although mTERF contacts were localized only at the boundary between the 16S rRNA/tRNA(Leu)UUR genes, multiple mtTFA contacts were detected. Contact sites were located in the light and the heavy strand promoters and, in agreement with in vitro footprinting data on human mitochondria, between the conserved sequence blocks (CSB) 1 and 2 and inside CSB-1. Potassium permanganate footprinting allowed detection of a 25-base pair region entirely contained in CSB-1 in which both strands were permanganate-reactive. No permanganate reactivity was associated with the other regions of the D-loop, including CSB-2 and -3, and with the mTERF contact site. We hypothesize that the single-stranded DNA at CSB-1 may be due to a profound helix distortion induced by mtTFA binding or be associated with a RNA polymerase pause site. In any case the location in CSB-1 of the 3' end of the most abundant replication primer and of the 5' end of the prominent D-loop DNA suggests that protein-induced DNA conformational changes play an important role in directing the transition from transcription to replication in mammalian mitochondria.

• Vijayasarathy C, Zheng YM, Mullick J, Basu A, Avadhani NG
• Identification of a stable RNA encoded by the H-strand of the mouse mitochondrial D-loop region and a conserved sequence motif immediately upstream of its polyadenylation site.
• Gene Expr. 1995; 4: 125-41
• Display abstract

• By using a combination of Northern blot hybridization with strand-specific DNA probes, S1 nuclease protection, and sequencing of oligo-dT-primed cDNA clones, we have identified a 0.8 kb poly(A)-containing RNA encoded by the H-strand of the mouse mitochondrial D-loop region. The 5' end of the RNA maps to nucleotide 15417, a region complementary to the start of tRNA(Pro) gene and the 3' polyadenylated end maps to nucleotide 16295 of the genome, immediately upstream of tRNA(Phe) gene. The H-strand D-loop region encoded transcripts of similar size are also detected in other vertebrate systems. In the mouse, rat, and human systems, the 3' ends of the D-loop encoded RNA are preceded by conserved sequences AAUAAA, AAUUAA, or AACUAA, that resemble the polyadenylation signal. The steady-state level of the RNA is generally low in dividing or in vitro cultured cells, and markedly higher in differentiated tissues like liver, kidney, heart, and brain. Furthermore, an over 10-fold increase in the level of this RNA is observed during the induced differentiation of C2C12 mouse myoblast cells into myotubes. These results suggest that the D-loop H-strand encoded RNA may have yet unknown biological functions. A 20 base pair DNA sequence from the 3' terminal region containing the conserved sequence motif binds to a protein from the mitochondrial extracts in a sequence-specific manner. The binding specificity of this protein is distinctly different from the previously characterized H-strand DNA termination sequence in the D-loop or the H-strand transcription terminator immediately downstream of the 16S rRNA gene. Thus, we have characterized a novel poly(A)-containing RNA encoded by the H-strand of the mitochondrial D-loop region and also identified the putative ultimate termination site for the H-strand transcription.

• Antoshechkin I, Bogenhagen DF
• Distinct roles for two purified factors in transcription of Xenopus mitochondrial DNA.
• Mol Cell Biol. 1995; 15: 7032-42
• Display abstract

• Transcription of Xenopus laevis mitochondrial DNA (xl-mtDNA) by the mitochondrial RNA polymerase requires a dissociable factor. This factor was purified to near homogeneity and identified as a 40-kDa protein. A second protein implicated in the transcription of mtDNA, the Xenopus homolog of the HMG box protein mtTFA, was also purified to homogeneity and partially sequenced. The sequence of a cDNA clone encoding xl-mtTFA revealed a high degree of sequence similarity to human and Saccharomyces cerevisiae mtTFA. xl-mtTFA was not required for basal transcription from a minimal mtDNA promoter, and this HMG box factor could not substitute for the basal factor, which is therefore designated xl-mtTFB. An antibody directed against the N terminus of xl-mtTFA did not cross-react with xl-mtTFB. xl-mtTFA is an abundant protein that appears to have at least two functions in mitochondria. First, it plays a major role in packaging mtDNA within the organelle. Second, DNase I footprinting experiments identified preferred binding sites for xl-mtTFA within the control region of mtDNA next to major mitochondrial promoters. We show that binding of xl-mtTFA to a site separating the two clusters of bidirectional promoters selectively stimulates specific transcription in vitro by the basal transcription machinery, comprising mitochondrial RNA polymerase and xl-mtTFB.

• Ghivizzani SC, Madsen CS, Nelen MR, Ammini CV, Hauswirth WW
• In organello footprint analysis of human mitochondrial DNA: human mitochondrial transcription factor A interactions at the origin of replication.
• Mol Cell Biol. 1994; 14: 7717-30
• Display abstract

• Using in organello footprint analysis, we demonstrate that within human placental mitochondria there is a high level of protein-DNA binding at regularly phased intervals throughout a 500-bp region encompassing the D-loop DNA origins and two promoter regions. Comparison with in vitro DNase I protection studies indicates that this protein-DNA interaction is due to non-sequence-specific binding by human mitochondrial transcription factor A (h-mtTFA). Since h-mtTFA can bend and wrap DNA, like its yeast counterpart ABF2, a primary function of h-mtTFA appears to be specific packaging of the mitochondrial DNA control region in vivo. Intervals of protein binding coincide with the spacing of the RNA start sites and prominent D-loop DNA 5' ends, suggesting a role for phased h-mtTFA binding in defining transcription and H-strand DNA replication origins. Significant protein-DNA interaction was also observed within the human homolog of conserved sequence block 1, both in organello and in vitro, using purified h-mtTFA.

• Lu T, Sawadogo M
• Role of the leucine zipper in the kinetics of DNA binding by transcription factor USF.
• J Biol Chem. 1994; 269: 30694-700
• Display abstract

• USF is a transcription factor characterized by a helix-loop-helix (HLH) DNA-binding domain that has been highly conserved through evolution. Vertebrate USFs contain an additional C-terminal leucine zipper (LZ) immediately adjacent to the HLH domain. This LZ is essential for efficient DNA binding by human USF. However, sea urchin has a USF family member that lacks a LZ and yet binds DNA efficiently. To clarify the role of the LZ in DNA binding by USF, we compared the properties of human and sea urchin USFs and found that the two proteins interacted with their specific sites on the DNA with identical affinities but very different kinetics. Association and dissociation rate constants of sea urchin USF were about 10-fold those of human USF. Domain-swapping experiments revealed that the LZ was responsible for the slower kinetics of human USF. USF heterodimers containing a single LZ displayed rates intermediate between those of dimers containing either two or no LZ, indicating that zipper-zipper interactions within USF dimers were not important for DNA binding. Temperature effects on DNA-binding parameters revealed a very high energy barrier for binding of human USF to DNA. Presence of a LZ increased the activation energy of the reaction.

• Polosa PL, Roberti M, Mustich A, Gadaleta MN, Cantatore P
• Purification and characterization of a mitochondrial DNA-binding protein that binds to double-stranded and single-stranded sequences of Paracentrotus lividus mitochondrial DNA.
• Curr Genet. 1994; 25: 350-6
• Display abstract

• A mitochondrial protein, able to specifically bind two double-stranded homologous sequences of sea-urchin mitochondrial DNA, has been partially purified from Paracentrotus lividus eggs. This protein, present at a low concentration, is a polypeptide of 40 kDa. One of the binding sequences, located in the main non-coding region, contains the replication origin of the mitochondrial DNA H-strand. By a combination of band-shift, DNase footprinting, and modification interference analyses with homologous and heterologous probes we identified YCYYATCAN(A/T)RC as the minimum sequence required for the binding. The protein also shows a single-stranded DNA-binding activity, as it is able to specifically interact with one of the strands of the binding sites. These features are consistent with a function of the protein in the modulation of sea-urchin mitochondrial DNA replication during the development stages.

• Qureshi SA, Jacobs HT
• Characterization of a high-affinity binding site for a DNA-binding protein from sea urchin embryo mitochondria.
• Nucleic Acids Res. 1993; 21: 811-6
• Display abstract

• Based on electrophoretic mobility shift assays, DNase I footprinting and modification interference analyses we have identified a sequence-specific DNA-binding protein in blastula stage mitochondria of the sea urchin Strongylocentrotus purpuratus, which interacts with a binding site around the major pause site for DNA replication. This region straddles the boundary of the genes for ATP synthase subunit 6 and cytochrome c oxidase subunit III, and contains also a prominent origin of lagging-strand synthesis. The protein is thermostable, and its natural high-affinity binding site comprises the sequence 5'-AGCCT(N7)AGCAT-3'. Binding studies have demonstrated that two copies of the imperfect repeat, as well as the 7 bp spacing between them, are essential for tight binding. Based on the location of its binding site, we tentatively designate the protein mitochondrial pause-region binding protein (mtPBP) 1.

• Qureshi SA, Jacobs HT
• Two distinct, sequence-specific DNA-binding proteins interact independently with the major replication pause region of sea urchin mtDNA.
• Nucleic Acids Res. 1993; 21: 2801-8
• Display abstract

• We have identified a second DNA-binding protein in sea urchin embryo mitochondria, which interacts with a binding site in the major replication pause region, at the junction of the genes for ATP synthase subunit 6 and cytochrome c oxidase subunit III (COIII). We provisionally designate this protein mtPBP2, to distinguish it from the previously characterized mitochondrial pause-region binding protein mtPBP1, whose properties and binding site are quite distinct. The high-affinity binding site for mtPBP2 lies at the 5' end of the COIII gene, and exhibits partial dyad symmetry, although modification interference analysis indicates that recognition is complex. Binding of mtPBP2 to this site induces a bend of approximately 45 degrees in the DNA. Southwestern blots show that mtPBP1 and 2 are both single polypeptides, of apparent molecular weights 25 kD and 18 kD respectively. In vitro, mtPBP1 and mtPBP2 bind independently to their high-affinity sites, which are separated by about 50 bp.

• Smith MJ, Arndt A, Gorski S, Fajber E
• The phylogeny of echinoderm classes based on mitochondrial gene arrangements.
• J Mol Evol. 1993; 36: 545-54
• Display abstract

• Previous analyses have demonstrated that, among the echinoderms, the sea star (class: Asteroidea) mitochondrial genome contains a large inversion in comparison to the mitochondrial DNA of sea urchins (class: Echinoidea). Polymerase chain reaction amplification, DNA cloning, and sequencing have been used to examine the relationships of the brittle stars (class: Ophiuroidea) and sea cucumbers (class: Holothuroidea) to the sea stars and sea urchins. The DNA sequence of the regions spanning potential inversion junctions in both brittle stars and sea cucumbers has been determined. This study has also revealed a highly modified tRNA cluster in the ophiuroid mitochondrial genome. Our data indicate mitochondrial gene arrangement patterns that group the sea cucumbers with sea urchins and sea stars with brittle stars. This use of molecular characters clarifies the relationships among these classes.

• Daga A, Micol V, Hess D, Aebersold R, Attardi G
• Molecular characterization of the transcription termination factor from human mitochondria.
• J Biol Chem. 1993; 268: 8123-30
• Display abstract

• The transcription termination factor (mTERF), which plays a central role in the control of mitochondrial rRNA and mRNA synthesis in mammalian mitochondria, has been previously identified and purified by DNA affinity chromatography from a human mitochondrial lysate (Kruse, B., Narasimhan, N., and Attardi, G. (1989) Cell 58, 391-397). In the present work, this factor has been characterized as to its protein composition and the activities of the protein components. Three polypeptides, two of approximately 34-kDa molecular mass and one of approximately 31 kDa, were shown to be associated with the specific DNA binding and footprinting activity of the factor, with the 31-kDa component having a much lower affinity for the recognition sequence than the 34-kDa components. On the other hand, the transcription termination activity, as assayed in an in vitro system, was found to be associated exclusively with the two 34-kDa polypeptides. Mass spectroscopic analysis of tryptic peptides derived from highly purified polypeptides indicated that all three polypeptides share regions with common sequences. The evidence obtained suggests that differential phosphorylation is not responsible for the difference in electrophoretic mobility of the three polypeptides.

• Roberti M, Mustich A, Gadaleta MN, Cantatore P
• Identification of two homologous mitochondrial DNA sequences, which bind strongly and specifically to a mitochondrial protein of Paracentrotus lividus.
• Nucleic Acids Res. 1991; 19: 6249-54
• Display abstract

• Using a combination of band shift and DNasel protection experiments, two Paracentrotus lividus mitochondrial sequences, able to bind tightly and selectively to a mitochondrial protein from sea urchin embryos, have been found. The two sequences, which compete with each other for binding to the protein, are located in two genome regions which are thought to contain regulatory signals for mitochondrial replication and transcription. A computer analysis suggests that the sequence TTTTRTANNTCYYATCAYA, common to the two binding regions, is the minimal recognition signal for the binding to the protein. We discuss the hypothesis that the protein binding capacity of these two sequences is involved in the control of sea urchin mtDNA replication during developmental stages.

• Jacobs HT, Herbert ER, Rankine J
• Sea urchin egg mitochondrial DNA contains a short displacement loop (D-loop) in the replication origin region.
• Nucleic Acids Res. 1989; 17: 8949-65
• Display abstract

• Based on solution hybridization using single-stranded probes, native mitochondrial DNA extracted from sea urchin eggs contains a displacement-loop (D-loop) of approximately 70-80 nt. This maps to the single extended unassigned sequence of the genome, between the genes for tRNA(thr) and tRNA(pro), which also appears to contain the origin of first-strand replication. The D-loop commences at or close to a site of supercoil-dependent S1 nuclease hypersensitivity, adjacent to a run of 20 consecutive C residues, terminates near to the boundary of tRNA(thr), and appears to be composed at least partly of RNA, based on the sensitivity of the assays to RNase H. These experiments imply that the mechanisms of replication initiation in sea urchin and vertebrate mtDNAs are very similar, and suggest that the developmental restriction on mtDNA synthesis in eggs and embryos is maintained at the level of D-loop extension.

• Mignotte B, Delain E, Rickwood D, Barat-Gueride M
• The Xenopus laevis mitochondrial protein mtDBP-C cooperatively folds the DNA in vitro.
• EMBO J. 1988; 7: 3873-9
• Display abstract

• The binding of the Xenopus laevis mitochondrial protein mtDBP-C to DNA was studied by equilibrium density banding, agarose gel electrophoresis and electron microscopy. The results obtained show that the mtDBP-C binds cooperatively to DNA irrespective of whether the DNA is supercoiled, relaxed or linear and it induces the formation of superhelical turns locally leading to the formation of a highly folded structure. It appears that this protein could be involved in the compaction of DNA in the mitochondrial nucleoid.

• Del Prete G, Minervini GR, Greco M, Gadaleta MN
• [In vivo synthesis of mitochondrial DNA during embryonal development of Paracentrotus lividus]
• Boll Soc Ital Biol Sper. 1980; 56: 1024-30
• Display abstract

• In vivo labeling of mitochondrial DNA using 3H-methyl-thymidine suggests that in the sea urchin Paracentrotus lividus also no bulk replication of mitochondrial DNA takes place after fertilization at least until pluteus stage.

• Lowell C, Bogenhagen D, Clayton DA
• S1 nuclease-specific nicking of mitochondrial DNA containing displacement loops.
• Anal Biochem. 1978; 91: 521-31
• Display abstract

• Conditions are described in which the single strand-specific nuclease S1 selectively nicks mitochondrial DNA containing displacement loops without nicking supercoiled mitochondrial DNA. Using these conditions, the percentage of molecules containing displacement loops can be easily and accurately determined. This method is superior to the traditional electron microscopic examination for assessing the frequency of displacement loop-containing molecules. In addition, this method permits the determination of the relative specific activities of displacement loop and nondisplacement loop-containing mitochondrial DNA after various radioactive labeling protocols. S1 nuclease is shown to cleave the displaced strand of the displacement loop, to partially degrade the 7S-initiation strand, but not to cleave the parental template strand complementary to the 7S-initiation strand. The final product is a nicked circular molecule with at least two breaks localized within the displacement loop region in only one of the two parental strands.

© 2021 EMBL | Privacy Policy