Search PubMed⌕ Search

Biomedical subjects

N C Martin

Publications and source records attributed to N C Martin.

At least 55 records · Page 3Linked to original sources
PubMed1991

RNase P RNA in Candida glabrata mitochondria is transcribed with substrate tRNAs.

The biosynthesis of some mitochondrial enzymes requires contributions of both the mitochondrial and nuclear genomes. The ribonucleoprotein enzyme Ribonuclease P (RNase P) is composed of a mitochondrial encoded RNA and nuclear coded protein in many yeasts, including C. glabrata. We have determined that there are at least two sites of transcription initiation that contribute to the expression of the mitochondrial RNase P RNA. A nonanucleotide promoter sequence is located upstream of the initiator tRNA while the other site of initiation of transcription is at an undetermined upstream site. An analysis of the transcripts from the region of the RNase P gene demonstrates directly that the RNase P RNA is present in large primary transcripts and located between the precursors to the initiator tRNAf(Met) and tRNA(Pro) genes. Thus this enzyme subunit is synthesized with some of its substrate tRNAs. An activity with cleavage site specificity like a previously described endonuclease that cleaves near the 3' end of tRNAs, RNase P activity and one or more additional endonucleases or exonucleases not described previously are required to convert the primary transcript to its final functional RNAs.

Base Sequence↗
PubMed1991

mRNA leader length and initiation codon context determine alternative AUG selection for the yeast gene MOD5.

MOD5, a nuclear gene of Saccharomyces cerevisiae, encodes two isozymic forms of a tRNA-modification enzyme. These enzymes modify both cytoplasmic and mitochondrial tRNAs. Two inframe ATGs of the MOD5 gene are used for initiation of translation, and the form of the protein translated from the first AUG is imported into mitochondria. Protein translated from the second AUG functions in the cytoplasm. Since all transcripts contain both of these translational start sites and two proteins are made, the question arises as to the factors that influence the translation start-site choice. Extending the 5' ends of the MOD5 mRNA to include leader sequences of the ADH1 (alcohol dehydrogenase defective) transcript produces significant changes in the choice of AUGs. This suggests that for wild-type MOD5 transcripts, the length or structure of the leader sequence plays a role in AUG choice. The nucleotides surrounding the first ATG of MOD5 also have an effect on translation initiation. Altering these nucleotides changes initiation choice and suggests that ribosomal bypass of a suboptimal AUG is another mechanism controlling the alternate use of two initiation codons. Our data support the model that at least one MOD5 transcript is able to produce two proteins with different N-terminal sequences.

Alkyl and Aryl Transferases↗
PubMed1991

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small.

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.

Base Sequence↗
PubMed1991

A gene required for RNase P activity in Candida (Torulopsis) glabrata mitochondria codes for a 227-nucleotide RNA with homology to bacterial RNase P RNA.

We have mapped a gene in the mitochondrial DNA of Candida (Torulopsis) glabrata and shown that it is required for 5' end maturation of mitochondrial tRNAs. It is located between the tRNAfMet and tRNAPro genes, the same tRNA genes that flank the mitochondrial RNase P RNA gene in the yeast Saccharomyces cerevisiae. The gene is extremely AT rich and codes for AU-rich RNAs that display some sequence homology with the mitochondrial RNase P RNA from S. cerevisiae, including two regions of striking sequence homology between the mitochondrial RNAs and the bacterial RNase P RNAs. RNase P activity that is sensitive to micrococcal nuclease has been detected in mitochondrial extracts of C. glabrata. An RNA of 227 nucleotides that is one of the RNAs encoded by the gene that we mapped cofractionated with this mitochondrial RNase P activity on glycerol gradients. The nuclease sensitivity of the activity, the cofractionation of the RNA with activity, and the homology of the RNA with known RNase P RNAs lead us to propose that the 227-nucleotide RNA is the RNA subunit of the C. glabrata mitochondrial RNase P enzyme.

Base Sequence↗
PubMed1991

MOD5 translation initiation sites determine N6-isopentenyladenosine modification of mitochondrial and cytoplasmic tRNA.

MOD5 is one of several genes that code for enzymes found in mitochondria and another cellular compartment. Like other such genes, it contains two in-frame ATGs that could be used to produce two proteins, differing from each other by an amino-terminal extension. Certain other genes produce heterogeneous mRNAs with some 5' ends falling upstream of the longest open reading frame and some 5' ends falling between the first and second ATGs. In these cases, selection of transcription start sites appears to play a significant role in translation start site selection. MOD5, in contrast, produces mRNAs with 5' ends that all fall upstream of both ATGs. To determine how MOD5 encodes isozymes that are located in different cellular compartments and to determine whether they differ in structure, we constructed MOD5 and MOD5-COXIV fusions with mutations of the first, second, or both ATGs. The effect of these alterations on protein production, tRNA modification, and cellular location was assessed. Both the first and second ATGs are used to produce MOD5 protein in vivo, but only the long form of the protein is imported into mitochondria. Thus, the first 11 amino acids present on the amino-terminal extended protein are necessary for mitochondrial import. Surprisingly, this extension does not promote complete import of the long form of the protein, but rather a functional pool of the extended protein remains in the cytoplasm. The amino-terminal extension is also unusual because it is probably not proteolytically removed upon import and therefore does not constitute part of a mitochondrial presequence.

Alkyl and Aryl Transferases↗
PubMed1990

Purification and properties of yeast ATP (CTP):tRNA nucleotidyltransferase from wild type and overproducing cells.

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) has been purified from wild type cells of the yeast Saccharomyces cerevisiae, as well as from a strain that overproduces the activity. Purification from the wild type strain was accomplished with a multistep protocol including ammonium sulfate fractionation, anion exchange chromatography, gel filtration, and affinity chromatography. The purified enzyme is near homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at 59,000 Da is smaller than reported previously. A similar molecular mass is obtained by gel filtration demonstrating that the enzyme is active as a monomer. The pH optimum for the enzyme is around 9.5. The apparent KM values for ATP and CTP were determined to be 5.6 x 10(-4) M and 1.8 x 10(-4) M, respectively. Purification of the enzyme from the overproducing cells was accomplished by a three step protocol with high yield. The nucleotidyltransferase activity from the overproducing cells had a KM for CTP indistinguishable from that of the wild type enzyme, and the mobility of the protein on sodium dodecyl sulfate gels was the same regardless of the source. Thus, the overproducing strain appears to be a good source for large amounts of yeast nucleotidyltransferase for further biochemical and structural studies.

Chromatography↗
PubMed1990

Isolation of a temperature-sensitive mutant with an altered tRNA nucleotidyltransferase and cloning of the gene encoding tRNA nucleotidyltransferase in the yeast Saccharomyces cerevisiae.

We have isolated a yeast mutant, ts352, that is temperature-sensitive for growth. The mutation has a general effect on mRNA metabolism and a specific effect on tRNA biosynthesis. Cells shifted to the nonpermissive temperature accumulate tRNAs that are shorter than mature tRNAs. The increased ability of these tRNAs to accept ATP demonstrates that growth of the ts352 mutant at the nonpermissive temperature results in accumulation of tRNA with defective 3' ends. The activity of ATP (CTP):tRNA-specific tRNA nucleotidyltransferase can readily be measured in extracts from wild type but not mutant cells. We have cloned and sequenced the wild type allele of the ts352 gene and find significant similarity between the yeast protein sequence predicted from the DNA sequence and the protein predicted from the sequence of the Escherichi coli tRNA nucleotidyltransferase gene. Expression of the yeast gene on a multicopy plasmid increases the activity of the tRNA nucleotidyltransferase in extracts. We conclude that the defect in the ts352 mutant is in the gene coding for yeast tRNA nucleotidyltransferase and that we have isolated the yeast gene that codes for this enzyme.

Amino Acid Sequence↗
PubMed1989

Characterization of yeast mitochondrial RNase P: an intact RNA subunit is not essential for activity in vitro.

We have previously described a mitochondrial activity that removes 5' leaders from yeast mitochondrial precursor tRNAs and suggested that it is a mitochondrial RNase P. Here we demonstrate that the cleavage reaction results in a 5' phosphate on the tRNA product and thus the activity is analogous to that of other RNase Ps. A mitochondrial gene called the tRNA synthesis locus encodes an A + U-rich RNA required for this activity in vivo. Two regions of this RNA display sequence similarity to conserved sequences in bacterial RNase P RNAs. This sequence similarity coupled with the analogous activities of the enzymes has led us to conclude that the RNAs are homologous and that the tRNA synthesis locus does code for the mitochondrial RNase P RNA subunit. The smallest and most abundant transcript of the tRNA synthesis locus is 490 nucleotides long. However, during purification of the holoenzyme, RNA is degraded and pieces of the original RNA are sufficient to support RNase P activity in vitro.

Bacillus subtilis↗
PubMed1989

N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.

Amino Acid Sequence↗
PubMed1989

A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice.

Erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located far upstream of the human beta-globin locus were inserted immediately upstream of a 4.1-kb fragment containing the human beta-globin gene. These constructs (HS beta) and a construct containing the beta-globin gene alone (beta) were microinjected into fertilized mouse eggs, and expression was analyzed in erythroid fetal liver and brain of day-16 embryos that developed. Only 7 of 23 animals that contained the beta gene alone expressed human beta-globin mRNA in erythroid tissue, and the average level of expression per gene copy was 0.3% of endogenous mouse beta-globin mRNA. In contrast, 50 of 51 transgenic mice that contained various HS beta constructs expressed the transgene specifically in erythroid tissue. The average level of expression per gene copy for constructs containing all five upstream HS sites was 109% of endogenous mouse beta-globin mRNA. Constructs that contained a single super-hypersensitive site (HS II beta) expressed 40% as much human beta-globin as mouse beta-globin mRNA per gene copy. These results demonstrate that the HS VI site, normally located downstream of the human beta-globin locus, is not required for high-level expression. Furthermore, the results demonstrate that high levels of human beta-globin gene expression can be obtained in transgenic mice even when a relatively small fragment of DNA (1.9 kb) containing erythroid-specific super-hypersensitive site II (HS II) is inserted upstream of the human beta-globin gene.

Animals↗
PubMed1989

Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

Amino Acid Sequence↗
PubMed1988

Biosynthesis of tRNA in yeast mitochondria. An endonuclease is responsible for the 3'-processing of tRNA precursors.

To study the mechanism involved in the 3'-processing of mitochondrial tRNA precursors, we examined tRNA processing in a reconstituted system with a yeast mitochondrial extract. Two mitochondrial tRNA(Glu) precursors synthesized from SP6 RNA polymerase-directed transcription system were used as substrates. One contained a 214-nucleotide 5' terminus and 115-123-nucleotide 3' trailer. The other had the same sized 3' trailer, but contained a mature 5' terminus. An endonucleolytic activity was identified in a mitochondrial S30 fraction which cleaves the 3' terminus of the latter tRNA precursor precisely at the in vivo CCA addition site. No cleavage of the 5'-extended precursor was observed in vitro. This mitochondrial 3'-processing activity was partially purified using DEAE-CL-6B chromatography. It removes the 3' trailer sequence from the 5'-matured precursor leaving a 3'-hydroxyl group on the processed tRNA and a 5'-phosphate group on the trailer. The resulting tRNA product serves as a substrate for tRNA nucleotidyltransferase which catalyzes the addition of CCA residues to the tRNA to complete its 3' maturation. Thus, yeast mitochondrial 3'-tRNA processing events resemble those found in eucaryotic cytoplasmic/nuclear systems where a single endonucleolytic cleavage is responsible for the formation of the 3' end of the tRNAs. This is in contrast to the multistep 3'-processing events known to occur in procaryotes.

Base Sequence↗
PubMed1987

Alteration of a mitochondrial tRNA precursor 5' leader abolishes its cleavage by yeast mitochondrial RNase P.

A mitochondrial specific RNase P is required to process 5' leaders from mitochondrial tRNA precursors in Saccharomyces cerevisiae. Experiments with a pair of mitochondrial pretRNAs(Asp) having leaders of different base composition suggest that this enzyme is unexpectedly sensitive to leader sequence or structure. Asp-AU (75% AU leader) is cleaved by the mitochondrial RNase P while Asp-GC (39% AU) is not. Both are substrates for E. coli RNase P. Partial nuclease digestions show that the tRNA portions of the two precursors differ in tertiary structure, while their 5' leaders differ in secondary structure. It is unusual for an RNaseP to have substrate specificity requirements which preclude processing of a pretRNA known to be a suitable substrate for an RNaseP from another species.

Bacterial Proteins↗
PubMed1987

Amino-terminal extension generated from an upstream AUG codon is not required for mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferase.

The TRM1 gene of Saccharomyces cerevisiae is necessary for the N2,N2-dimethylguanosine modification of both mitochondrial and cytoplasmic tRNAs. The DNA sequence of the TRM1 locus and the 5' ends of mRNAs expressed from this gene have been determined. The majority of the 5' ends map within a large open reading frame between two in-frame ATGs at positions +1 and +49. A small fraction of the 5' ends are located upstream of the first ATG. Both AUGs of the TRM1 mRNAs are used to initiate translation, and two forms of N2,N2-dimethylguanosine-specific tRNA methyltransferase, which differ by an amino-terminal extension of 16 amino acids, are made. Mitochondrial tRNAs are modified when the initiation of translation is limited to one or the other of the AUGs, suggesting that the amino-terminal extension is not necessary for import of the protein into mitochondria. Mitochondrial targeting information must, therefore, be located in a region of N2,N2-dimethylguanosine-specific tRNA methyltransferase that is found in both forms of the enzyme.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of MOD5, a gene required for isopentenylation of cytoplasmic and mitochondrial tRNAs of Saccharomyces cerevisiae.

The mod5-1 mutation is a nuclear mutation in Saccharomyces cerevisiae that reduces the biosynthesis of N6-(delta 2-isopentenyl)adenosine in both cytoplasmic and mitochondrial tRNAs to less than 1.5% of wild-type levels. The tRNA modification enzyme, delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase, cannot be detected in vitro with extracts from mod5-1 cells. A characterization of the MOD5 gene would help to determine how the same enzyme activity in different cellular compartments can be abolished by a single nuclear mutation. To that end we have cloned the MOD5 gene and shown that it restores delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase activity and N6-(delta 2-isopentenyl)adenosine to tRNA in both the mitochondria and the nucleus/cytoplasm compartments of mod5-1 yeast cells. That MOD5 sequences are expressed in Escherichia coli and can complement an N6-(delta 2-isopentenyl)-2-methylthioadenosine-deficient E. coli mutant leads us to conclude that MOD5 is the structural gene for delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase.

Alkyl and Aryl Transferases↗
PubMed1987

DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts.

A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.

Alkyl and Aryl Transferases↗