@article {1167, title = {Initiation of mitochondrial DNA replication by transcription and R-loop processing.}, journal = {The Journal of Biological Chemistry}, volume = {273}, year = {1998}, month = {1998 Nov 13}, pages = {30614-21}, abstract = {

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.

}, keywords = {Base Sequence, Catalytic Domain, Cell Line, Circular, DNA, DNA Replication, Endoribonucleases, Genetic, Humans, Mitochondrial, Molecular Sequence Data, Nucleic Acid Conformation, Ribonucleoproteins, RNA, Transcription}, issn = {0021-9258}, doi = {10.1101/gad.1352105}, author = {Lee, D Y and Clayton, D A} } @article {1186, title = {Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice.}, journal = {Nature Genetics}, volume = {18}, year = {1998}, month = {1998 Mar}, pages = {231-6}, abstract = {

The regulation of mitochondrial DNA (mtDNA) expression is crucial for mitochondrial biogenesis during development and differentiation. We have disrupted the mouse gene for mitochondrial transcription factor A (Tfam; formerly known as m-mtTFA) by gene targetting of loxP-sites followed by cre-mediated excision in vivo. Heterozygous knockout mice exhibit reduced mtDNA copy number and respiratory chain deficiency in heart. Homozygous knockout embryos exhibit a severe mtDNA depletion with abolished oxidative phosphorylation. Mutant embryos proceed through implantation and gastrulation, but die prior to embryonic day (E)10.5. Thus, Tfam is the first mammalian protein demonstrated to regulate mtDNA copy number in vivo and is essential for mitochondrial biogenesis and embryonic development.

}, keywords = {Animals, Developmental, DNA, DNA-Binding Proteins, Embryo Implantation, Female, Fetal Death, Fetal Growth Retardation, Gene Dosage, Gene Expression Regulation, Heart, Heterozygote, High Mobility Group Proteins, Integrases, Knockout, Mice, Mitochondria, Mitochondrial, Mitochondrial Proteins, Mutation, Nuclear Proteins, Phosphorylation, Transcription Factors, Viral Proteins}, issn = {1061-4036}, doi = {10.1038/ng0398-231}, author = {Larsson, N G and Wang, J and Wilhelmsson, H and Oldfors, A and Rustin, P and Lewandoski, M and Barsh, G S and Clayton, D A} } @article {1187, title = {RNase MRP correctly cleaves a novel R loop at the mitochondrial DNA leading-strand origin of replication.}, journal = {Genes \& Development}, volume = {11}, year = {1997}, month = {1997 Mar}, pages = {582-92}, abstract = {

The precursor primer RNA for mammalian mitochondrial DNA leading-strand replication remains as a persistent R loop formed during transcription through the mitochondrial DNA control region. We have examined model R loops, which exist in a novel and physiologically accurate preprimer conformation, as potential substrates for mammalian RNase mitochondrial RNA processing (MRP). Mouse RNase MRP accurately cleaves an R loop containing the mouse mitochondrial DNA origin. The multiple cleavage sites on the R-loop substrate match the priming sites observed in vivo, suggesting that RNase MRP alone is capable of generating virtually all of the leading-strand replication primers.

}, issn = {0890-9369}, author = {Clayton, D A and Lee, D Y} } @article {1169, title = {Addition of a 29 residue carboxyl-terminal tail converts a simple HMG box-containing protein into a transcriptional activator.}, journal = {Journal of Molecular Biology}, volume = {249}, year = {1995}, month = {1995 May 26}, pages = {11-28}, abstract = {

Human mitochondrial transcription factor A (h-mtTFA) is essential for initiation of transcription from the two promoters located in the displacement-loop region of human mitochondrial DNA. This 25 kDa protein contains two tandem, HMG box DNA-binding domains separated by a 27 amino acid residue linker region and followed by a 25 residue carboxyl-terminal tail; both the linker and tail are rich in basic amino acid residues. Mutational analysis of h-mtTFA revealed that the tail region is important for specific DNA recognition and essential for transcriptional activation. The critical role of the human tail in transcription was confirmed by constructing chimeric proteins that exchanged similar regions between h-mtTFA and its Saccharomyces cerevisiae homolog, sc-mtTFA. Wild-type sc-mtTFA is unable to activate transcription from the human mitochondrial light-strand promoter (LSP). Addition of the human tail region to sc-mtTFA conferred LSP-specific promoter activation. In all of the different h-mtTFA mutations tested, transcriptional activation was correlated with specific DNA-binding activity, suggesting that these two functions may be inseparable, a situation entirely consistent with previous mutational analyses of human mitochondrial promoters.

}, keywords = {Base Sequence, DNA, DNA-Binding Proteins, Genetic, High Mobility Group Proteins, Humans, Mitochondrial, Mitochondrial Proteins, Molecular Sequence Data, Mutagenesis, Nuclear Proteins, Promoter Regions, Saccharomyces cerevisiae, Site-Directed, Trans-Activators, Transcription Factors}, issn = {0022-2836}, doi = {10.1101/gad.1352105}, author = {Dairaghi, D J and Shadel, G S and Clayton, D A} } @article {1170, title = {A persistent RNA-DNA hybrid is formed during transcription at a phylogenetically conserved mitochondrial DNA sequence.}, journal = {Molecular and Cellular Biology}, volume = {15}, year = {1995}, month = {1995 Jan}, pages = {580-9}, abstract = {

Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA hybrid formation that begins at the 5{\textquoteright} end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable hybrids, and the presence of rGs within the RNA strand of the RNA-DNA hybrid is required. The efficiency of hybrid formation depends on the length of RNA synthesized 5{\textquoteright} to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA hybrid.

}, keywords = {Base Sequence, DNA, DNA Primers, DNA Replication, DNA-Directed DNA Polymerase, DNA-Directed RNA Polymerases, Genetic, Mitochondrial, Molecular Sequence Data, Nucleic Acid, Nucleic Acid Hybridization, Phylogeny, Regulatory Sequences, Ribonuclease H, RNA, Saccharomyces cerevisiae, Transcription}, issn = {0270-7306}, doi = {10.1101/gad.1352105}, author = {Xu, B and Clayton, D A} } @article {1171, title = {A human mitochondrial transcriptional activator can functionally replace a yeast mitochondrial HMG-box protein both in vivo and in vitro.}, journal = {Molecular and Cellular Biology}, volume = {13}, year = {1993}, month = {1993 Mar}, pages = {1951-61}, abstract = {

Human mitochondrial transcription factor A is a 25-kDa protein that binds immediately upstream of the two major mitochondrial promoters, thereby leading to correct and efficient initiation of transcription. Although the nature of yeast mitochondrial promoters is significantly different from that of human promoters, a potential functional homolog of the human transcriptional activator protein has been previously identified in yeast mitochondria. The importance of the yeast protein in yeast mitochondrial DNA function has been shown by inactivation of its nuclear gene (ABF2) in Saccharomyces cerevisiae cells resulting in loss of mitochondrial DNA. We report here that the nuclear gene for human mitochondrial transcription factor A can be stably expressed in yeast cells devoid of the yeast homolog protein. The human protein is imported efficiently into yeast mitochondria, is processed correctly, and rescues the loss-of-mitochondrial DNA phenotype in a yeast abf2 strain, thus functionally substituting for the yeast protein. Both human and yeast proteins affect yeast mitochondrial transcription initiation in vitro, suggesting that the two proteins may have a common role in this fundamental process.

}, keywords = {Amino Acid, Amino Acid Sequence, Base Sequence, Biological Transport, Cell Compartmentation, DNA-Binding Proteins, Genes, Genetic, Humans, Mitochondria, Mitochondrial Proteins, Molecular Sequence Data, Nuclear Proteins, Oxygen Consumption, Post-Translational, Protein Processing, Protein Sorting Signals, Saccharomyces cerevisiae, Sequence Alignment, Sequence Homology, Transcription, Transcription Factors, Transformation}, issn = {0270-7306}, doi = {10.1101/gad.1352105}, author = {Parisi, M A and Xu, B and Clayton, D A} } @article {1173, title = {Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies.}, journal = {Nature}, volume = {351}, year = {1991}, month = {1991 May 16}, pages = {236-9}, abstract = {

Defects in mitochondrial DNA (mtDNA) are associated with several different human diseases, including the mitochondrial encephalomyopathies. The mutations include deletions but also duplications and point mutations. Individuals with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) carry a common A-to-G substitution in a highly conserved portion of the gene for transfer RNA(Leu(UUR)). Although the MELAS mutation may be comparable to the defect in the tRNA(Lys) gene associated with MERRF (myoclonus epilepsy associated with ragged-red fibres), it is also embedded in the middle of a tridecamer sequence necessary for the formation of the 3{\textquoteright} ends of 16S ribosomal RNA in vitro. We found that the MELAS mutation results in severe impairment of 16S rRNA transcription termination, which correlates with a reduced affinity of the partially purified termination protein for the MELAS template. This suggests that the molecular defect in MELAS is the inability to produce the correct type and quantity of rRNA relative to other mitochondrial gene products.

}, keywords = {Base Sequence, Blotting, Brain Diseases, Chromosome Mapping, DNA, DNA Mutational Analysis, Genetic, Humans, Mitochondria, Mitochondrial, Molecular Sequence Data, Muscle, Mutation, Peptide Termination Factors, Ribosomal, RNA, Transcription, Transfer, Western}, issn = {0028-0836}, doi = {10.1101/gad.1352105}, author = {Hess, J F and Parisi, M A and Bennett, J L and Clayton, D A} } @article {1172, title = {Similarity of human mitochondrial transcription factor 1 to high mobility group proteins.}, journal = {Science}, volume = {252}, year = {1991}, month = {1991 May 17}, pages = {965-9}, abstract = {

Human mitochondrial transcription factor 1 (mtTF1) has been sequenced and is a nucleus-encoded DNA binding protein of 204 amino acids (24,400 daltons). Expression of human mtTF1 in bacteria yields a protein with correct physical properties and the ability to activate mitochondrial DNA promoters. Analysis of the protein{\textquoteright}s sequence reveals no similarities to any other DNA binding proteins except for the existence of two domains that are characteristic of high mobility group (HMG) proteins. Human mtTF1 is most closely related to a DNA binding HMG-box region in hUBF, a human protein known to be important for transcription by RNA polymerase I.

}, keywords = {Amino Acid Sequence, Base Sequence, Blotting, DNA, DNA-Binding Proteins, Gene Library, Genetic, High Mobility Group Proteins, Humans, Messenger, Mitochondria, Mitochondrial, Mitochondrial Proteins, Molecular Sequence Data, Molecular Weight, Northern, Nuclear Proteins, Nucleic Acid, Nucleic Acid Hybridization, Oligonucleotide Probes, Open Reading Frames, Promoter Regions, RNA, Sequence Homology, Transcription Factors}, issn = {0036-8075}, doi = {10.1101/gad.1352105}, author = {Parisi, M A and Clayton, D A} } @article {1174, title = {Flexible recognition of rapidly evolving promoter sequences by mitochondrial transcription factor 1.}, journal = {Genes \& Development}, volume = {3}, year = {1989}, month = {1989 Dec}, pages = {2202-17}, abstract = {

Transcriptional promoters of mitochondrial DNA have diverged extensively in the course of mammalian evolution. Nevertheless, the transcriptional machinery and the overall mechanisms of transcriptional control and regulation seem to be conserved. We have compared the human and murine homologs of the major DNA-binding transcriptional activator, mitochondrial transcription factor 1 (mtTF1), with unexpected results. Both proteins have similar chromatographic and transcriptional properties and are the same size. Both recognize and bind sequences between -12 and -39 within their respective homologous promoters. However, the sequences that they recognize are markedly divergent; although the base pairs they contact are situated similarly or identically with respect to the transcriptional start site, sequence identity between the two species{\textquoteright} contact points is less than 50\%. Interestingly, the two proteins are functionally interchangeable; each can bind to the heterologous light-strand promoter and can activate transcription by the heterologous mitochondrial RNA polymerase. Thus, the RNA polymerase or some as yet undetected transcription factor, rather than mTF1, may determine the strict species specificity of mitochondrial transcription. Flexible DNA sequence recognition by mtTF1, on the other hand, may be a principal facilitating mechanism for rapid control sequence evolution.

}, keywords = {Animals, Base Sequence, Chromatography, DNA, Genetic, Humans, Ion Exchange, Methylation, Mice, Mitochondrial, Molecular Sequence Data, Nucleic Acid, Promoter Regions, Sequence Homology, Transcription, Transcription Factors}, issn = {0890-9369}, doi = {10.1101/gad.1352105}, author = {Fisher, R P and Parisi, M A and Clayton, D A} } @article {1175, title = {Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate.}, journal = {Cell}, volume = {56}, year = {1989}, month = {1989 Jan 13}, pages = {131-9}, abstract = {

Using complementary oligonucleotide probes, we have isolated the nuclear gene for the RNA moiety of RNAase MRP; it is present as a single copy and encodes an uncapped primary transcript of 275 nucleotides. Direct sequence analysis revealed that the 136 nucleotide RNA that copurifies with RNAase MRP represents the 3{\textquoteright} half of the 275 nucleotide primary transcript. The 5{\textquoteright}-flanking region of the gene has putative transcriptional control elements homologous to the promoters of RNA polymerase II-transcribed U-series snRNA genes; however, the coding region possesses a box A sequence and terminates at four T residues, both features characteristic of polymerase III-transcribed genes. A decamer sequence, 5{\textquoteright}-CGA-CCCCUCC-3{\textquoteright}, complementary to a conserved sequence adjacent to the enzymatic cleavage site on the mitochondrial RNA substrate, is present in the RNAase MRP RNA. Isolation of a nuclear gene for the RNA component of a mitochondrial enzyme implies that nucleic acids can be transported across mitochondrial membranes.

}, keywords = {Animals, Blotting, DNA, Endoribonucleases, Genes, Genetic, Mice, Mitochondria, Mitochondrial, Nucleic Acid, Oligonucleotide Probes, Post-Transcriptional, Ribonucleases, RNA Processing, Sequence Homology, Southern, Transcription}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Chang, D D and Clayton, D A} } @article {1178, title = {A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA.}, journal = {Science}, volume = {235}, year = {1987}, month = {1987 Mar 6}, pages = {1178-84}, abstract = {

Ribonuclease mitochondrial RNA processing, a site-specific endoribonuclease involved in primer RNA metabolism in mammalian mitochondria, requires an RNA component for its activity. On the basis of copurification and selective inactivation with complementary oligonucleotides, a 135-nucleotide RNA species, not encoded in the mitochondrial genome, is identified as the RNA moiety of the endoribonuclease. This finding implies transport of a nucleus-encoded RNA, essential for organelle DNA replication, to the mitochondrial matrix.

}, keywords = {Animals, Base Sequence, Cell Nucleus, Chemical Phenomena, Chemistry, Drug Resistance, Endonucleases, Enzyme Activation, Genetic Code, Humans, Mammals, Micrococcal Nuclease, Mitochondria, Oligonucleotides, Organoids, Ribonucleases, RNA, Subcellular Fractions}, issn = {0036-8075}, doi = {10.1101/gad.1352105}, author = {Chang, D D and Clayton, D A} } @article {1177, title = {Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.}, journal = {Cell}, volume = {50}, year = {1987}, month = {1987 Jul 17}, pages = {247-58}, abstract = {

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

}, keywords = {DNA, DNA-Directed RNA Polymerases, Genes, Genetic, Humans, KB Cells, Mitochondria, Mitochondrial, Promoter Regions, Regulator, Templates, Transcription, Transcription Factors}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Fisher, R P and Topper, J N and Clayton, D A} } @article {1176, title = {Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7.}, journal = {Cell}, volume = {51}, year = {1987}, month = {1987 Oct 9}, pages = {89-99}, abstract = {

Analysis of the nucleotide sequence of the genetic locus for yeast mitochondrial RNA polymerase (RPO41) reveals a continuous open reading frame with the coding potential for a polypeptide of 1351 amino acids, a size consistent with the electrophoretic mobility of this enzymatic activity. The transcription product from this gene spans the singular reading frame. In vivo transcript abundance reflects codon usage and growth under stringent conditions for mitochondrial biogenesis and function results in a several fold higher level of gene expression than growth under glucose repression. A comparison of the yeast mitochondrial RNA polymerase amino acid sequence to those of E. coli RNA polymerase subunits failed to demonstrate any regions of homology. Interestingly, the mitochondrial enzyme is highly homologous to the DNA-directed RNA polymerases of bacteriophages T3 and T7, especially in regions most highly conserved between the T3 and T7 enzymes themselves.

}, keywords = {Amino Acid Sequence, Base Sequence, DNA, DNA-Directed RNA Polymerases, Escherichia coli, Genetic, Mitochondria, Molecular Sequence Data, Nucleic Acid, Saccharomyces cerevisiae, Sequence Homology, T-Phages, Transcription}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Masters, B S and Stohl, L L and Clayton, D A} } @article {1179, title = {DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity.}, journal = {Cell}, volume = {45}, year = {1986}, month = {1986 Jun 20}, pages = {817-25}, abstract = {

DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex.

}, keywords = {Centrifugation, Density Gradient, DNA, DNA Primase, Endoribonucleases, Humans, Micrococcal Nuclease, Mitochondria, Mitochondrial, Ribonuclease H, Ribosomal, RNA, RNA Nucleotidyltransferases}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Wong, T W and Clayton, D A} } @article {1180, title = {In vitro replication of human mitochondrial DNA: accurate initiation at the origin of light-strand synthesis.}, journal = {Cell}, volume = {42}, year = {1985}, month = {1985 Oct}, pages = {951-8}, abstract = {

Synthesis of human light-strand mitochondrial DNA was accomplished in vitro using DNA primase, DNA polymerase, and other accessory proteins isolated from human mitochondria. Replication begins with the synthesis of primer RNA on a T-rich sequence in the origin stem-loop structure of the template DNA and absolutely requires ATP. A transition from RNA synthesis to DNA synthesis occurs near the base of the stem-loop structure and a potential recognition site for signaling that transition has been identified. The start sites of the in vitro products were mapped at the nucleotide level and were found to be in excellent agreement with those of in vivo nascent light-strand DNA. Isolated human mitochondrial enzymes recognize and utilize the bovine, but not the mouse, origin of light-strand replication.

}, keywords = {Animals, Base Sequence, Cattle, Cloning, DNA, DNA Replication, DNA Restriction Enzymes, Genetic, Humans, KB Cells, Macromolecular Substances, Mitochondrial, Molecular, Nucleic Acid Conformation, Templates}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Wong, T W and Clayton, D A} } @article {1182, title = {Replication priming and transcription initiate from precisely the same site in mouse mitochondrial DNA.}, journal = {The EMBO Journal}, volume = {4}, year = {1985}, month = {1985 Jun}, pages = {1559-67}, abstract = {

Mammalian mitochondrial DNA maintains a novel displacement-loop region containing the major sites of transcriptional initiation and the origin of heavy strand DNA replication. Because the exact map positions of the 5{\textquoteright} termini of nascent mouse displacement-loop strands are known, it is possible to examine directly a potential relationship between replication priming and transcription. Analyses of in vivo nucleic acids complementary to the displacement-loop region reveal two species with identical 5{\textquoteright} ends at map position 16 183. One is entirely RNA and the other is RNA covalently linked to DNA. In the latter the transition from RNA to DNA is sharp, occurring near or within a series of previously identified conserved sequences 74-163 nucleotides downstream from the transcriptional initiation site. These data suggest that the initial events in replication priming and transcription are the same and that the decision to synthesize DNA or RNA is a downstream event under the control of short, conserved displacement-loop template sequences.

}, keywords = {Animals, Base Sequence, Chromosome Mapping, DNA, DNA Replication, Genetic, Mice, Mitochondrial, Operon, Ribonucleases, RNA, Transcription}, issn = {0261-4189}, doi = {10.1101/gad.1352105}, author = {Chang, D D and Hauswirth, W W and Clayton, D A} } @article {1181, title = {A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro.}, journal = {The Journal of Biological Chemistry}, volume = {260}, year = {1985}, month = {1985 Sep 15}, pages = {11330-8}, abstract = {

Faithful transcription of human mitochondrial DNA has been reproduced in vitro, using a fraction of mitochondrial proteins capable of accurate initiation at both the heavy- and light-strand promoters. Here we report the initial dissection of this system into two nonfunctional components which, upon mixing, reconstitute promoter-specific transcriptional capacity in vitro. One of these components copurifies with the major nonspecific RNA polymerase activity, suggesting its identity. The other component lacks significant polymerase activity, but contains a protein or proteins required for accurate initiation at the two individual promoters by isolated mitochondrial RNA polymerase. This factor facilitates specific transcription, but has little or no effect on nonspecific transcription of a synthetic copolymer (poly(dA-dT)), indicating a positive role in proper promoter recognition. The transcription factor markedly stimulates light-strand transcription, but only moderately enhances transcription initiation at the heavy-strand promoter, suggesting different or additional factor requirements for heavy-strand transcription. These requirements may reflect the functional differences between heavy- and light-strand transcription in vivo and, in particular, the role of the light-strand promoter in priming of heavy-strand DNA replication.

}, keywords = {Carcinoma, Cell Line, DNA-Directed RNA Polymerases, Genetic, Humans, Kinetics, Messenger, Mitochondria, Mouth Neoplasms, Promoter Regions, RNA, Templates, Transcription, Transcription Factors}, issn = {0021-9258}, doi = {10.1101/gad.1352105}, author = {Fisher, R P and Clayton, D A} } @article {1183, title = {Precise identification of individual promoters for transcription of each strand of human mitochondrial DNA.}, journal = {Cell}, volume = {36}, year = {1984}, month = {1984 Mar}, pages = {635-43}, abstract = {

The major site of in vivo transcriptional initiation for both heavy and light strands of human mitochondrial DNA is the displacement-loop region. Transcripts synthesized in vitro by human mitochondrial RNA polymerase were mapped to the nucleotide level and have identical 5{\textquoteright} end map positions to those reported for in vivo primary transcripts. An ordered series of deletion clones, whose template sequences were truncated at either the 5{\textquoteright} or 3{\textquoteright} end, was used to identify the precise mitochondrial DNA sequence required for initiation of transcription. The data provide a definitive assignment of the promoter for heavy-strand transcription occurring within -16 to +7 of the transcriptional start site 16 nucleotides upstream of the 5{\textquoteright} end of the gene for tRNAPhe and of the promoter for light-strand transcription occurring within -28 to +16 of the transcriptional start site at the 5{\textquoteright} end of "7S RNA." Within each control sequence is a candidate promoter whose consensus sequence is 5{\textquoteright}-CANACC(G)CC(A)AAAGAPyA-3{\textquoteright} and in both cases transcriptional initiation occurs within six to eight nucleotides of the 3{\textquoteright} end of this sequence. The transcriptional start site is an integral part of each promoter and each promoter can function in the absence of the other.

}, keywords = {Chromosome Deletion, Chromosome Mapping, DNA, DNA-Directed RNA Polymerases, Genes, Genetic, Humans, Mitochondrial, Operon, Regulator, RNA, Transcription, Transfer}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Chang, D D and Clayton, D A} } @article {1184, title = {Sequence and gene organization of mouse mitochondrial DNA.}, journal = {Cell}, volume = {26}, year = {1981}, month = {1981 Oct}, pages = {167-80}, abstract = {

The complete sequence of the 16,295 bp mouse L cell mitochondrial DNA genome has been determined. Genes for the 12S and 16S ribosomal RNAs; 22 tRNAs; cytochrome c oxidase subunits I, II and III; ATPase subunit 6; cytochrome b; and eight unidentified proteins have been located. The genome displays exceptional economy of organization, with tRNA genes interspersed between rRNA and protein-coding genes with zero or few noncoding nucleotides between coding sequences. Only two significant portions of the genome, the 879 nucleotide displacement-loop region containing the origin of heavy-strand replication and the 32 nucleotide origin of light-strand replication, do not encode a functional RNA species. All of the remaining nucleotide sequence serves as a defined coding function, with the exception of 32 nucleotides, of which 18 occur at the 5{\textquoteright} ends of open reading frames. Mouse mitochondrial DNA is unique in that the translational start codon is AUN, with any of the four nucleotides in the third position, whereas the only translational stop codon is the orthodox UAA. The mouse mitochondrial DNA genome is highly homologous in overall sequence and in gene organization to human mitochondrial DNA, with the descending order of conserved regions being tRNA genes; origin of light-strand replication; rRNA genes; known protein-coding genes; unidentified protein-coding genes; displacement-loop region.

}, keywords = {Animals, Base Sequence, Chromosome Mapping, Codon, Cytochromes, DNA, Genes, L Cells (Cell Line), Mice, Mitochondrial, NADH Dehydrogenase, Protein Biosynthesis, Ribosomal, RNA, Transfer}, issn = {0092-8674}, doi = {10.1101/gad.1352105}, author = {Bibb, M J and Van Etten, R A and Wright, C T and Walberg, M W and Clayton, D A} } @article {1185, title = {Circular dimer and catenate forms of mitochondrial DNA in human leukaemic leucocytes.}, journal = {Nature}, volume = {216}, year = {1967}, month = {1967 Nov 18}, pages = {652-7}, keywords = {Centrifugation, Chemical Phenomena, Chemistry, Chromatography, Cytogenetics, Density Gradient, DNA, Electron, Humans, Ion Exchange, Leukemia, Microscopy, Mitochondria, Molecular Weight, Neoplasm}, issn = {0028-0836}, doi = {10.1101/gad.1352105}, author = {Clayton, D A and Vinograd, J} }