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D A Clayton

Publications and source records attributed to D A Clayton.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of a Xenopus laevis ribonucleoprotein endoribonuclease. Isolation of the RNA component and its expression during development.

In order to facilitate studies of the assembly and transport of the site-specific RNase mitochondrial RNA processing (MRP) ribonucleoprotein, we have characterized it from Xenopus laevis cells. X. laevis RNase MRP displayed a similar spectrum of cleavage activity to that produced by previously isolated mammalian nuclear enzymes. A 277-nucleotide RNA component of the ribonucleoprotein was identified; the gene for the RNA was isolated, sequenced, and found to be 66 and 63% similar to mouse and human RNase MRP RNAs, respectively. Despite the evolutionary distance from its mammalian counterparts, X. laevis RNase MRP RNA contains five regions of homology to the mammalian RNase MRP RNA. Four of these regions correspond to those previously identified as conserved between RNase MRP and RNase P RNAs; the fifth encompasses nucleotides recently discovered to be sufficient for autoantigen binding. The expression and assembly of Xenopus RNase MRP RNA were examined in frog oocytes and developing embryos. RNase MRP RNA was expressed throughout oogenesis; it started to accumulate at stage I and reached a maximum in stage IV. During embryogenesis RNase MRP RNA expression began to elevate at approximately stage 22 and continued to rise through the swimming tadpole stage. When injected into the nucleus of mature oocytes, the X. laevis RNase MRP RNA gene was expressed accurately, and transcripts were packaged into immunoprecipitable particles.

Animals↗
PubMed1992

Assignment of a yeast protein necessary for mitochondrial transcription initiation.

Yeast mitochondrial DNA contains multiple promoters that are responsible for expression of its genes. The basic yeast mitochondrial promoter consists of a nonanucleotide consensus sequence [5'-ATATAAGTA(+1)-3'] that must be recognized by transcription proteins, including mitochondrial RNA polymerase and any relevant trans-acting factors. Since mitochondrial RNA polymerase alone appeared unable to recognize a mitochondrial promoter, we examined the effects of providing accessory proteins to enable promoter function. After expression in Escherichia coli or purification from yeast mitochondria, two proteins were tested; they were ABF2 (a structural homologue of the human mitochondrial transcriptional activator mtTF1) and MTF1 (the gene product of a yeast locus known to exhibit a mitochondrial transcription phenotype). The results show that MTF1 specifies correct transcriptional initiation while ABF2 does not. We conclude that MTF1 is an essential key protein in yeast mitochondrial promoter function. Considering the increasing complexity of the mitochondrial transcription apparatus, we propose a nomenclature system for its components.

Base Sequence↗
PubMed1992

DNA wrapping and bending by a mitochondrial high mobility group-like transcriptional activator protein.

Mitochondrial transcription factor 1 (mtTF1) is the only accessory protein known to be required for accurate and efficient promoter recognition by mammalian mitochondrial RNA polymerase. It activates transcription by binding immediately upstream of transcriptional start sites and shows an inherent flexibility in primary DNA sequence requirement. By application of a purification strategy designed for human and mouse mtTF1, a protein resembling mtTF1 was recently isolated from yeast mitochondria; its size (19 kDa), DNA-binding properties, and amino acid composition suggest identity to HM, a previously described abundant protein of yeast mitochondria. Both human and yeast proteins show a general ability to wrap or condense and unwind DNA in vitro and bend DNA at specific sequences. Recent determinations of the amino acid sequences of the human and yeast proteins reveal that both contain domains homologous to the nuclear high mobility group (HMG) proteins which have been implicated in diverse functions such as chromatin compaction and transcription stimulation. The ability to unwind and bend DNA may be fundamental to the documented roles of the mammalian protein in mitochondrial DNA transcription and replication priming and suggests a similar function for the yeast protein in yeast mitochondria.

Amino Acid Sequence↗
PubMed1992

Structure and function of the mitochondrial genome.

It is now clear that molecular defects in human mitochondrial DNA play a significant role in human disease. Mitochondrial DNA mutations range from single base changes in the 16.5 kilobase-pair genome up to large deletions and rearrangements. Independently of the actual cause of a given mutation, it is possible to predict at least some of the consequences of changes in mitochondrial DNA sequence. This paper reviews our overall understanding of the mode and mechanism of mitochondrial DNA replication and transcription and how this relates to mitochondrial gene expression. This provides a background to anticipate the nature and extent of mitochondrial DNA sequence changes that might be of physiological consequence.

Animals↗
PubMed1992

Transcription and replication of animal mitochondrial DNAs.

The development of in vitro transcription and replication systems has allowed the identification of promoter sequences and origins of replication for several animal mtDNAs. As a consequence, the necessary reagents and basic information are available to permit the characterization of transacting factors that are required for transcription and replication. All of the animal trans-acting species purified at this time are known or reasoned to be nuclear gene products. There is now the opportunity to learn how these nuclear genes are regulated and the mechanisms that are utilized for the import of their products into the organelle. With regard to import, the human transcription factor mtTF1 appears to have an amino-terminal sequence characteristic of other imported mitochondrial proteins (Parisi and Clayton, 1991). An interesting issue is the degree to which fundamental features of mtDNA replication and transcription are conserved between species. With regard to animal mtDNAs, there is very little in the way of conservation of DNA sequence at the promoters and origins of replication. The exceptions to this are the presence of a characteristic stem-loop L-strand origin of replication sequence in vertebrates [except for chicken mtDNA (Desjardins and Morais, 1990)] and the general presence of CSBs II and III (and to a lesser extent CSB I) in most higher animal mtDNAs. Because mtDNA promoters are not highly conserved, it is perhaps not surprising that general cross-species transcription does not occur, except for very limited examples of closely related species and sequences (Chang et al., 1985b). Using crude mtRNA polymerase holoenzyme preparations, there is no specific transcriptional initiation when proteins from human mitochondria are used with mouse mtDNA promoter templates, and vice versa. However, in contrast to this overall observation, purified fractions of human or mouse mtTF1 can be exchanged and shown to function across species boundaries (Fisher et al., 1989). The ability of mitochondrial mtTF1-type proteins to operate across even greater evolutionary distances was suggested by the ability of human and yeast proteins to recognize some mitochondrial promoter sequences in common (Fisher et al., 1992). More recent studies suggest that human mtTF1 can substitute for its yeast homolog in vivo, and thereby perform at least the most critical functions required to maintain yeast mtDNA in the cell (M.A. Parisi, B. Xu, and D.A. Clayton, submitted for publication). The other sites of conserved macromolecular interactions are related to the two origins of DNA replication.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Yeast site-specific ribonucleoprotein endoribonuclease MRP contains an RNA component homologous to mammalian RNase MRP RNA and essential for cell viability.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA sequence complementary to mammalian mitochondrial origins of replication in a manner consistent with a role in primer RNA metabolism. The same activity in the yeast Saccharomyces cerevisiae has recently been identified; it cleaves an RNA substrate complementary to a yeast mitochondrial origin of replication at an exact site of linkage of RNA to DNA. We have purified this yeast enzyme further and detect a single, novel RNA of 340 nucleotides associated with the enzymatic activity. The single-copy nuclear gene for this RNA was sequenced and mapped to the right arm of chromosome XIV. The identity of the clone, as encoding the RNA copurifying with enzymatic activity, was confirmed by a match to the directly determined sequence of the RNA. The gene sequence also identified a 340-nucleotide RNA in total yeast RNA and in purified RNase MRP enzyme preparations. Inspection of the sequence of the yeast RNA revealed homologies to the RNA component of mouse RNase MRP, 49% overall with specific regions of much greater similarity. The flanking regions of the gene showed characteristics of an RNA polymerase II transcription unit, including a TATAAA box and a 7/8 match to the yeast cell cycle box UAS. The RNase MRP RNA gene was deleted by insertional replacement and found to be essential for cellular viability, indicating a critical nuclear role for RNase MRP.

Animals↗
PubMed1992

Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

Base Sequence↗
PubMed1991

Similarity of human mitochondrial transcription factor 1 to high mobility group proteins.

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'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.

Amino Acid Sequence↗
PubMed1991

Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies.

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' 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.

Base Sequence↗
PubMed1991

A rapid, efficient method for purifying DNA-binding proteins. Denaturation-renaturation chromatography of human and yeast mitochondrial extracts.

We describe a novel method for the purification of DNA-binding proteins. Isolated mitochondria were lysed in boiling sodium dodecyl sulfate-containing buffer, the extracts were chromatographed on hydroxylapatite in the presence of sodium dodecyl sulfate, and DNA-binding activities were identified after adding a large excess of nonionic detergent (Triton X-100) and assaying fractions by a gel retardation procedure. Fractions containing DNA-binding activity were bulk renatured and chromatographed on phosphocellulose in the presence of Triton X-100. When applied to human mitochondria, the technique resulted in the purification to homogeneity of fully functional mitochondrial transcription factor 1 (mtTF1), the major activator of mammalian mitochondrial transcription. Moreover, the yield of mtTF1 purified by this method was at least 25 times higher than that obtained by conventional nondenaturing chromatographies. When yeast mitochondria were subjected to the same protein isolation scheme, a 19-kilodalton putative yeast homologue of mtTF1 was purified to homogeneity. These results suggest that the denaturation-renaturation approach may be a valuable general method for the identification and high yield purification of DNA-binding proteins.

Amino Acid Sequence↗
PubMed1991

Nuclear gadgets in mitochondrial DNA replication and transcription.

In mammalian mitochondrial DNA, activation of the light-strand promoter mediates both priming of leading-strand replication and initiation of light-strand transcription. Accurate and efficient transcription requires at least two proteins: mitochondrial RNA polymerase and a separable transcription factor that can function across species boundaries. Subsequently, primer RNAs are cleaved by a site-specific ribonucleoprotein endoribonuclease that recognizes short, highly conserved sequence elements in the RNA substrate.

Animals↗
PubMed1991

Nuclear RNase MRP processes RNA at multiple discrete sites: interaction with an upstream G box is required for subsequent downstream cleavages.

RNase MRP is a site-specific endoribonuclease that processes primer RNA from the leading-strand origin of mammalian mitochondrial DNA replication. It is present in active form as isolated from the nucleus, suggesting a bipartite cellular location and function. The relatively high abundance of nucleus-localized RNase MRP has permitted its purification to near homogeneity and, in turn, has led to the identification of protein components of this ribonucleoprotein. Analysis of the mode of RNA cleavage by nuclear RNase MRP revealed the surprising and unprecedented ability of the endonuclease to process RNA at multiple discrete locations. Substrate cleavage is dependent on the presence of a previously described G-rich sequence element adjacent to the primary site of RNA processing. Downstream cleavage occur in a distance- and sequence-specific manner.

Animals↗
PubMed1990

Secondary structure of the RNA component of a nuclear/mitochondrial ribonucleoprotein.

RNase mitochondrial RNA processing (MRP) is a site-specific endoribonuclease located in both the nucleus and mitochondria of vertebrate cells. The enzyme is a ribonucleoprotein whose RNA component has been shown to be encoded by a nuclear gene. Because RNase MRP is particular in its substrate requirement, RNA-RNA interaction has been proposed as important for the cleavage reaction. A secondary structure of this RNA from mouse cells has been derived by chemical modification of in vivo MRP RNA in ribonucleoprotein form, as isolated free RNA, and as RNA synthesized in vitro. Full-length MRP RNA appears to adopt a conformation containing a significant number of single-stranded residues and may form a pseudoknot. The data are consistent with both the RNA within the ribonucleoprotein and the free RNA possessing comparable secondary structures and suggest a possible site of interaction between enzyme and substrate. The human MRP RNA can be folded into a conformation very similar to that predicted for the mouse MRP RNA. A more limited analysis of human MRP RNA is consistent with the structure proposed for the mouse species.

Animals↗
PubMed1990

Characterization of human MRP/Th RNA and its nuclear gene: full length MRP/Th RNA is an active endoribonuclease when assembled as an RNP.

Vertebrate cells contain a site-specific endoribonuclease (RNase MRP) that cleaves mitochondrial RNA transcribed from the origin of leading-strand mitochondrial DNA replication. This report presents the characterization of the human enzyme and its essential RNA component. Human RNase MRP is a ribonucleoprotein with a nucleus-encoded RNA of 265 nucleotides. As expected, the single-copy RNA coding region is homologous (84%) to the corresponding mouse gene; surprisingly, at least 700 nucleotides of the immediate 5'-flanking region are conserved. The 265-nucleotide MRP RNA and an MRP RNA cleavage product representing the 3'-terminal 108 nucleotides exist in nuclear and mitochondrial RNA isolates; the larger MRP RNA is present in greatest abundance in the nucleus. The putative processing site within the 265-nucleotide MRP RNA is offset from that of mouse MRP RNA, but in each case cleavage is precise and occurs at the sequence ANCCCGC. Oligonucleotide-mediated inhibition experiments reveal that both the 5' and 3' portions of the MRP RNA are involved in cleavage by RNase MRP; this implies that full length MRP RNA complexed with proteins is an active species in vertebrate cells.

Animals↗
PubMed1990

The gene for the RNA component of the mitochondrial RNA-processing endoribonuclease is located on human chromosome 9p and on mouse chromosome 4.

Mitochondrial RNA-processing endoribonuclease (RNAase MRP) has the capacity to cleave mitochondrial RNA complementary to the light strand of the displacement loop at a unique site. The enzyme is a ribonucleoprotein whose RNA component is a nuclear gene product. The 5' flanking region of the primary transcript has control elements characteristic of RNA polymerase II transcription, and the coding region has features of RNA polymerase III transcription signals. The RNA associated with RNAase MRP is the first known RNA encoded by a single-copy gene in the nucleus and believed to be imported into mitochondria. The gene (RMRP) for this RNA component of RNAase MRP was assigned to human chromosome 9 and mouse chromosome 4 by Southern blot analyses of 11 human X rodent hybrids and 11 mouse X rodent hybrids with probe pHM1.0 and probe pSP270, respectively. In situ hybridization of probe pHSTU300 to normal human chromosomes revealed 29 of 100 cells with label on 9p and 9.6% of 302 silver grains located at 9p21--p12.

Animals↗
PubMed1990

Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.

Animals↗