Biomedical subjects
L A Grivell
Publications and source records attributed to L A Grivell.
Transcription of mitochondrial DNA.
While mitochondrial DNA (mtDNA) is the simplest DNA in nature, coding for rRNAs and tRNAs, results of DNA sequence, and transcript analysis have demonstrated that both the synthesis and processing of mitochondrial RNAs involve remarkably intricate events. At one extreme, genes in animal mtDNAs are tightly packed, both DNA strands are completely transcribed (symmetric transcription), and the appearance of specific mRNAs is entirely dependent on processing at sites signalled by the sequences of the tRNAs, which abut virtually every gene. At the other extreme, gene organization in yeast (Saccharomyces) is anything but compact, with long stretches of AT-rich DNA interspaced between coding sequences and no obvious logic to the order of genes. Transcription is asymmetric and several RNAs are initiated de novo. Nevertheless, extensive RNA processing occurs due largely to the presence of split genes. RNA splicing is complex, is controlled by both mitochondrial and nuclear genes, and in some cases is accompanied by the formation of RNAs that behave as covalently closed circles. The present article reviews current knowledge of mitochondrial transcription and RNA processing in relation to possible mechanisms for the regulation of mitochondrial gene expression.
Mosaic genes and RNA processing in mitochondria.
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Mitochondrial mosaics-maturases on the move.
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Isolation and characterization of nuclear genes coding for subunits of the yeast ubiquinol-cytochrome c reductase complex.
Nuclear genes coding for the Mr 17 000, 14 000 and 11 000 subunits of the ubiquinol-cytochrome c reductase complex (complex III) in yeast have been isolated from a clone bank of yeast nuclear DNA by use of a mRNA hybridization-competition assay. This is based on our observations that levels of mRNAs for these subunits are much reduced during glucose repression and in cytoplasmic petite mutants and the procedure should be of general application for the isolation of other inducible or repressible genes coding for mRNAs present at low levels in the cell. A first characterization of the clones is presented. The genes are not closely linked in the genome and those coding for Mr 14 000 and 11 000 subunits are present in unique genomic environments, which suggests that there are only single copies of each in the nuclear genome.
Small is beautiful--portrait of a mitochondrial genome.
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One gene's intron is another gene's exon.
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In vitro suppression of UGA codons in a mitochondrial mRNA.
Although both prokaryotic and eukaryotic messenger RNAs can be easily translated in heterologous protein-synthesizing systems, attempts to achieve correct synthesis of mitochondrial proteins by translation of mitochondrial mRNAs in such systems have failed. In general, the products of synthesis are of low molecular weight and presumably represent fragments of mitochondrial proteins. These fragments display a strong tendency to aggregate. Explanations have included the use by mitochondria of codons requiring a specialized tRNA population and the fortuitous occurrence within genes of purine-rich sequences resembling bacterial ribosome binding sites. In addition, the long 5'-leader sequences present in many mitochondrial (mt) RNAs may also contribute to difficulties in mRNA recognition by heterologous ribosomes. Recent sequence analysis of human mtDNA suggests that the genetic code used by mammalian mitochondria deviates in a number of respects from the 'universal' code, the most striking of these being the use of the UGA termination codon to specify tryptophan. That this may also apply in yeast mitochondria has been shown by Fox and Macino et al., thus providing an obvious and easily testable explanation for the inability of heterologous systems to synthesize full-length mitochondrial proteins. We confirm this explanation and describe here the in vitro synthesis of a full-length subunit II of yeast cytochrome c oxidase in a wheat-germ extract supplemented with a partially purified mitochondrial mRNA for this protein and a UGA-suppressor tRNA from Schizosaccharomyces pombe.
Mutations affecting RNA splicing and the interaction of gene expression of the yeast mitochondrial loci cob and oxi-3.
In Saccharomyces cerevisiae strains KL14-4A and 777-3A, four intervening sequences of 1900 (l alpha beta), 1400 (l beta gamma), 1300 (l gamma delta) and 650 bp (l delta epsilon) separate the five coding sequences (alpha-epsilon) of the structural gene (cob) for cytochrome b. Its major transcript is an 18S RNA (2200 nucleotides) which is likely to be the functional mRNA. The lengths of a series of larger transcripts and their hybridization with probes specific for different intervening sequences are consistent with their being intermediates in a splicing process which generates 18S RNA from a giant primary transcript (greater than or equal to 7.5 kb) covering the whole cob region. There is no absolute order of splicing. The intervening sequence l alpha beta is excised in two stages. The first generates a stable 10S RNA, coded for by sequences immediately downstream of the 18S RNA coding segment alpha. The function of this RNA is unknown. Its excision is an early step in processing, whereas excision of the remainder of l alpha beta is a late event. We have studied four cytochrome b-deficient mutants. These map in intervening sequences and are splicing-defective. They accumulate 22S-28S RNAs which contain one or more intervening sequences. The l alpha beta mutants synthesize long, novel polypeptides, antigenically related to cytochrome b, possibly as a result of read-through into the intervening sequences. Several cob mutants also display alterations in their transcripts of oxi-3, the locus which codes for cytochrome c oxidase subunit I. This indicates that interactions between cob and oxi-3 exist at the level of RNA processing.
Some yeast mitochondrial RNAs are circular.
11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.
Transcription maps of mtDNAs of two strains of saccharomyces: transcription of strain-specific insertions; Complex RNA maturation and splicing.
We have developed a two-dimensional method for simultaneously mapping on the yeast mtDNA genome all the transcripts representing more than 0.01% of mtRNA. In two yeast strains, Saccharomyces carlsbergensis NCYC-74 and Saccharomyces cerevisiae KL14-4A, about 25 discrete transcripts were found apart from tRNAs. The mtDNAs of these strains differ by the absence (NCYC-74) or presence (KL 14-4A) of various large insertions located within genetically active regions. The transcripts can all be related to known loci on the genetic map. In nearly all cases the RNAs are much longer than required to specify the known protein product of the locus concerned. The organization of the transcripts is similar in the two strains except at the positions of the large insertions (500-3300 bp) in the oxi-3 and cob loci. The sequences of these insertions are present in RNA species larger than 25S, but are absent from smaller transcripts of the same regions. This is probably due to splicing, since the coding sequences for most of these smaller transcripts are noncontiguous. The smaller transcripts of other loci also seem to arise from processing of larger RNA species. The oxi-3 locus, containing the structural gene for cytochrome c oxidase subunit l, is transcribed in a very complex fashion that suggests differential splicing into partially overlapping transcripts. This may indicate that oxi-3 has additional genetic functions, including possible control of the biosynthesis of cytochrome c oxidase holoenzyme or its assembly into the mitochondrial inner membrane. As in the case of the eucaryote nucleus, the regulation of mitochondrial gene expression seems to occur more at the level of RNA processing than has been recognized thus far.
Nucleotide sequence of the mitochondrial structural gene for subunit 9 of yeast ATPase complex.
We have determined the nucleotide sequence of a segment of Saccharomyces mtDNA that contains the structural gene for one of the subunits (the dicyclohexylcarbodiimide-binding protein) of the mitochondrial ATPase complex. The sequence fits the known amino acid sequence of this protein with the exception of one amino acid. Codon usage is biased in favor of A + T-rich codons. On both sides of the gene, the nucleotide sequence contains less than 4% (mol/mol) G + C for at least 180 nucleotides; these A + T sequences show no evidence of internal repetition. The gene and all the A + T-rich sequence preceding the gene are present in a 12S RNA that is the major transcript of this segment of mtDNA. The nature of the sequences responsible for binding ribosomes to mitochondrial mRNA and for termination of RNA synthesis is considered.
Identification of mitochondrial gene products by DNA-directed protein synthesis in vitro.
1. A cell-free system, derived from Escherichia coli is highly active in the linked transcription-translation of yeast mtDNA from both wild-type and petite strains. 2. The products of synthesis are short (Mr less than 10 000) hydrophobic polypeptides, which show a high tendency to aggregate in a specific fashion with E. coli and mitochondrial proteins. Aggregation is extremely persistent: alkali, sodium dodecyl sulphate/urea, guanidinium . HCl and carboxymethylation reduce it, but do not eliminate it completely. 3. Nevertheless, results of indirect immunoprecipitation tests suggest that antigenic determinants of cytochrome c oxidase are among the products synthesized. The immunoprecipitation appears specific by criteria including competition experiments and its absence when mtDNA from low complexity petites, retaining only the gene for 21 S rRNA and some flanking sequences, is used to programme protein synthesis. Electrophoretic analysis of material precipitated by anti-cytochrome c oxidase sera reveals four discrete polypeptides with molecular weights of 7400, 6400, 5000 and 4100, which probably represent polypeptide fragments carrying antigenic determinants of cytochrome c oxidase.
Transcription in yeast mitochondria: isolation and physical mapping of messenger RNAs for subunits of cytochrome c oxidase and ATPase.
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The mitochondrial genome of yeast.
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A coupled transcription--translation system derived from Escherichia coli: the use of immobilized deoxyribonuclease to eliminate endogenous DNA.
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Minimal post-transcriptional modification of yeast mitochondrial ribosomal RNA.
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Yeast mitochondrial RNA does not contain poly(A)
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