In vitro studies of disease-linked variants of human tRNA nucleotidyltransferase reveal decreased thermal stability and altered catalytic activity.

Mutations in the human TRNT1 gene encoding tRNA nucleotidyltransferase (tRNA-NT), an essential enzyme responsible for addition of the CCA (cytidine-cytidine-adenosine) sequence to the 3'-termini of tRNAs, have been linked to disease phenotypes including congenital sideroblastic anemia with B-cell immunodeficiency, periodic fevers and developmental delay (SIFD) or retinitis pigmentosa with erythrocyte microcytosis. The effects of these disease-linked mutations on the structure and function of tRNA-NT have not been explored. Here we use biochemical and biophysical approaches to study how five SIFD-linked amino acid substitutions (T154I, M158V, L166S, R190I and I223T), residing in the N-terminal head and neck domains of the enzyme, affect the structure and activity of human tRNA-NT in vitro. Our data suggest that the SIFD phenotype is linked to poor stability of the T154I and L166S variant proteins, and to a combination of reduced stability and altered catalytic efficiency in the M158 V, R190I and I223T variants.

The Saccharomyces cerevisiae enolase-related regions encode proteins that are active enolases.

In addition to two genes (ENO1 and ENO2) known to code for enolase (EC4.2.1.11), the Saccharomyces cerevisiae genome contains three enolase-related regions (ERR1, ERR2 and ERR3) which could potentially encode proteins with enolase function. Here, we show that products of these genes (Err2p and Err3p) have secondary and quaternary structures similar to those of yeast enolase (Eno1p). In addition, Err2p and Err3p can convert 2-phosphoglycerate to phosphoenolpyruvate, with kinetic parameters similar to those of Eno1p, suggesting that these proteins could function as enolases in vivo. To address this possibility, we overexpressed the ERR2 and ERR3 genes individually in a double-null yeast strain lacking ENO1 and ENO2, and showed that either ERR2 or ERR3 could complement the growth defect in this strain when cells are grown in medium with glucose as the carbon source. Taken together, these data suggest that the ERR genes in Saccharomyces cerevisiae encode a protein that could function in glycolysis as enolase. The presence of these enolase-related regions in Saccharomyces cerevisiae and their absence in other related yeasts suggests that these genes may play some unique role in Saccharomyces cerevisiae. Further experiments will be required to determine whether these functions are related to glycolysis or other cellular processes.

Analysis of the pathogenic I326T variant of human tRNA nucleotidyltransferase reveals reduced catalytic activity and thermal stability in vitro linked to a conformational change.

The I326T mutation in the TRNT1 gene encoding human tRNA nucleotidyltransferase (tRNA-NT) is linked to a relatively mild form of SIFD. Previous work indicated that the I326T variant was unable to incorporate AMP into tRNAs in vitro, however, expression of the mutant allele from a strong heterologous promoter supported in vivo CCA addition to both cytosolic and mitochondrial tRNAs in a yeast strain lacking tRNA-NT. To address this discrepancy, we determined the biochemical and biophysical characteristics of the I326T variant enzyme and the related variant, I326A. Our in vitro analysis revealed that the I326T substitution decreases the thermal stability of the enzyme and causes a ten-fold reduction in enzyme activity. We propose that the structural changes in the I326T variant that lead to these altered parameters result from a rearrangement of helices within the body domain of the protein which can be probed by the inability of the monomeric enzyme to form a covalent dimer in vitro mediated by C373. In addition, we confirm that the effects of the I326T or I326A substitutions are relatively mild in vivo by demonstrating that the mutant alleles support both mitochondrial and cytosolic CCA-addition in yeast.

Accurate transcription of a plant mitochondrial gene in vitro.

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

Mutational analysis of the Prt1 protein subunit of yeast translation initiation factor 3.

The Saccharomyces cerevisiae PRT1 gene product Prt1p is a component of translation initiation factor eIF-3, and mutations in PRT1 inhibit translation initiation. We have investigated structural and functional aspects of Prt1p and its gene. Transcript analysis and deletion of the PRT1 5' end revealed that translation of PRT1 mRNA is probably initiated at the second in-frame ATG in the open reading frame. The amino acid changes encoded by six independent temperature-sensitive prt1 mutant alleles were found to be distributed throughout the central and C-terminal regions of Prt1p. The temperature sensitivity of each mutant allele was due to a single missense mutation, except for the prt1-2 allele, in which two missense mutations were required. In-frame deletion of an N-terminal region of Prt1p generated a novel, dominant-negative form of Prt1p that inhibits translation initiation even in the presence of wild-type Prt1p. Subcellular fractionation suggested that the dominant-negative Prt1p competes with wild-type Prt1p for association with a component of large Prt1p complexes and as a result inhibits the binding of wild-type Prt1p to the 40S ribosome.

Two inactive fragments derived from the yeast mitochondrial ribosomal protein MrpS28 function in trans to support ribosome assembly and respiratory growth.

The mitochondrial ribosomal protein MrpS28 of Saccharomyces cerevisiae is one of several mitochondrial ribosomal proteins homologous to Escherichia coli ribosomal proteins within the context of a larger protein. Relative to a region of homology with E. coli ribosomal protein S15, the mature MrpS28 protein has unique sequence domains of 117 and 48 amino acids at its amino and carboxyl terminus, respectively. To better understand the role of the various sequence domains of the MrpS28 protein in vivo, truncated derivatives were expressed under conditions where they were the only potential source of functional MrpS28 protein. The results shown here demonstrate that the amino-terminal domain and the S15-like domain are both essential for respiratory growth. Interestingly an inactive amino-terminal fragment can be complemented in trans by a second inactive fragment comprising the S15-like domain and the carboxyl-terminal 48 amino acids. Consequently, the assembly of these fragments into ribosomal subunits can be examined when they are expressed individually or together. Results from these studies indicate that each of the MrpS28-derived fragments facilitates the incorporation of the other into 37 S ribosomal subunits.

Derivatives of the yeast mitochondrial ribosomal protein MrpS28 replace ribosomal protein S15 as functional components of the Escherichia coli ribosome.

The mitochondrial ribosomal protein MrpS28 is considerably larger than its eubacterial homolog, Escherichia coli ribosomal protein S15 (Eco S15). Relative to a region of homology that spans the entire length of the bacterial protein, mature MrpS28 is extended by 117 and 48 amino acids at its amino and carboxyl termini, respectively. Both the amino-terminal and S15-like domains of MrpS28 are essential for function in yeast mitochondria. Here, we show that these same two domains function in E. coli. The S15-like domain of MrpS28 alone complements a cold-sensitive mutation in E. coli strain KR121 that gives rise to reduced levels of Eco S15. However, complementation by the S15-like domain of MrpS28 is inefficient when compared with Eco S15. Surprisingly, the amino-terminal domain of MrpS28, which is apparently a unique component of the mitochondrial ribosome and is unable by itself to complement the cold-sensitive phenotype, enhances the ability of the S15-like domain to support growth of KR121 cells at nonpermissive temperatures. Together, these data suggest that the amino-terminal domain contributes to the fundamental properties of MrpS28 involved in the assembly and function of both mitochondrial and E. coli ribosomes.

Mapping CDC mutations in the yeast S. cerevisiae by rad52-mediated chromosome loss.

Using the chromosome loss-mapping method of Schild and Mortimer, I have mapped several new temperature-sensitive mutations that define five CDC genes. Modified procedures were used to facilitate mapping temperature-sensitive mutations in general, and these modifications are discussed. The mutations were assigned to specific chromosomes by chromosome loss procedures, and linkage relationships were determined subsequently by standard tetrad analysis. Four of the mutations define new loci. The fifth mutation, cdc63-1, is shown to be allelic to previously known mutations in the PRT1 gene.

A high-copy-number ADE2-bearing plasmid for transformation of Candida glabrata.

The Candida glabrata ADE2 gene encoding aminoimidazole ribonucleotide (AIR) carboxylase (EC 4.1.1.21) was isolated by complementation of the ade2-1 mutation in Saccharomyces cerevisiae. The predicted amino acid (aa) sequence is 75% identical to that of S. cerevisiae. Integrative transformation was used to produce a C. glabrata strain bearing a deletion of ADE2 coding sequences. A high-copy-number shuttle vector bearing the ADE2 gene was constructed and contains a fragment of S. cerevisiae mitochondrial (mt) DNA that confers the ability to replicate autonomously in C. glabrata.

Purification and characterization of a tRNA nucleotidyltransferase from Lupinus albus and functional complementation of a yeast mutation by corresponding cDNA.

ATP (CTP):tRNA nucleotidyltransferase (EC 2.7.7.25) was purified to apparent homogeneity from a crude extract of Lupinus albus seeds. Purification was accomplished using a multistep protocol including ammonium sulfate fractionation and chromatography on anion-exchange, hydroxylapatite and affinity columns. The lupin enzyme exhibited a pH optimum and salt and ion requirements that were similar to those of tRNA nucleotidyltransferases from other sources. Oligonucleotides, based on partial amino acid sequence of the purified protein, were used to isolate the corresponding cDNA. The cDNA potentially encodes a protein of 560 amino acids with a predicted molecular mass of 64 164 Da in good agreement with the apparent molecular mass of the pure protein determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The size and predicted amino acid sequence of the lupin enzyme are more similar to the enzyme from yeast than from Escherichia coli with some blocks of amino acid sequence conserved among all three enzymes. Functionality of the lupin cDNA was shown by complementation of a temperature-sensitive mutation in the yeast tRNA nucleotidyltransferase gene. While the lupin cDNA compensated for the nucleocytoplasmic defect in the yeast mutant it did not enable the mutant strain to grow at the non-permissive temperature on a non-fermentable carbon source.

Processing of transfer RNA precursors in a wheat mitochondrial extract.

To investigate mechanisms for processing of plant mitochondrial RNAs, we studied the fate of wheat mitochondrial tRNA precursors in a homologous soluble extract. Artificial precursor transcripts were synthesized in vitro using T3 or T7 RNA polymerase and DNA templates containing wheat mitochondrial tRNA genes and flanking sequences. We found that the mitochondrial extract supports processing of precursors containing both native and chloroplast-like (Joyce, P. B. M., and Gray, M. W. (1989) Nucleic Acids Res. 77, 5461-5476) wheat mitochondrial tRNA sequences. Incubation of precursor transcripts with the extract results in processing of tRNAs via precise 5'- and 3'-endonucleolytic cleavages. However, these cleavages are not ordered in vitro because intermediates composed of 5'-leader + tRNA and tRNA + 3'-trailer are present simultaneously throughout the course of the reaction. Sequence analysis of processed products confirmed that endonucleolytic cleavages occur at the expected positions, generating tRNAs with 5'-phosphoryl and 3'-hydroxyl termini. The mitochondrial extract also contains a tRNA nucleotidyltransferase activity that adds -CCAOH termini to the 3'-ends of processed tRNAs. This cell-free RNA processing system provides the basis for biochemical characterization of the various enzymes involved in the production and maturation of plant mitochondrial tRNAs.

Molecular characterization of the yeast PRT1 gene in which mutations affect translation initiation and regulation of cell proliferation.

Several temperature-sensitive cell division cycle (cdc) mutations differentially affect the regulatory step for cell proliferation in the yeast Saccharomyces cerevisiae. We recently found that one of these mutations, cdc63-1, resides in a gene called PRT1; other mutations in this gene had been previously shown to affect translation initiation. Here we report the molecular cloning and characterization of the PRT1 gene from yeast. Our results show that the PRT1 gene is an essential, single-copy gene which encodes a 2500-nucleotide polyadenylated transcript. The nucleotide sequence indicates that the gene could code for a protein product of Mr 88,000, which bears no overall amino acid sequence similarity to any other known protein but which contains similarity over a limited region to amino acid sequences involved in nucleotide binding.

Regulated arrest of cell proliferation mediated by yeast prt1 mutations.

Several temperature-sensitive cell-division-cycle (cdc) mutations differentially affect the regulatory step for cell proliferation in the yeast. Saccharomyces cerevisiae, including one mutation termed cdc63-1, which resides in a previously known gene called PRT1. Other mutations in the PRT1 gene have been shown by others to affect an initiation step in protein synthesis. Here we show that at the appropriate nonpermissive temperature each prt1 mutation can produce a uniform and concerted arrest of cell division; the prt1-1 mutation, like cdc63-1, is shown to arrest cells specifically at the regulatory step for cell proliferation. This response of cessation of cell division is different from the response of cells to an equivalent limitation of protein synthesis using cycloheximide or verrucarin A, which implies that the PRT1 gene product could separately influence both cellular growth via protein synthesis and events in the regulation of cell proliferation.

Isolation and nucleotide sequence of a gene encoding tRNA nucleotidyltransferase from Kluyveromyces lactis.

A gene (KlCCA1) encoding ATP(CTP):tRNA specific tRNA nucleotidyltransferase (EC 2.7.7.25) was isolated from Kluyveromyces lactis by complementation of the Saccharomyces cerevisiae cca1-1 mutation. Sequencing of a 2665 bp EcoRI-SpeI restriction fragment revealed an open reading frame potentially encoding a protein of 489 amino acids with 57% sequence similarity to its S. cerevisiae homologue. Southern hybridization revealed a single copy of KlCCA1 in the K. lactis genome. KlCCA1 was able to complement both the mitochondrial and cytosolic defects in the cca1-1 mutant, suggesting that, as in S. cerevisiae, the K. lactis gene encodes a sorting isozyme that is targeted to mitochondria and the nucleus and/or cytosol. An altered KlCCA1 gene encoding a tRNA nucleotidyltransferase that lacked its first 35 amino acids was able to complement the nuclear/cytosolic but not the mitochondrial defect in the S. cerevisiae cca1-1 mutant, suggesting that the 35 amino-terminal amino acids are necessary for targeting to mitochondria but are not required for enzyme activity. Our results suggest that the mechanisms for production and distribution of mitochondrial and nuclear/cytosolic tRNA nucleotidyltransferase in K. lactis differ from those seen in S. cerevisiae.

In vitro processing of transcripts containing novel tRNA-like sequences ('t-elements') encoded by wheat mitochondrial DNA.

We have recently described the properties of a wheat mitochondrial extract that is able to process, accurately and efficiently, artificial transcripts containing wheat mitochondrial tRNA sequences, with the production of mature tRNAs (P.J. Hanic-Joyce and M.W. Gray, J. Biol. Chem., in press). Such processing involves 5'-endonucleolytic, 3'-endonucleolytic, and tRNA nucleotidyltransferase activities. Here we show that this system also acts on transcripts containing sequences corresponding to an unusual class of short repeats ('t-elements') in wheat mtDNA. These repeats are theoretically capable of assuming a tRNA-like secondary structure, although stable transcripts corresponding to them are not detectable in vivo. We find that t-element sequences are processed with the same specificity and with comparable efficiency as are authentic tRNA sequences. Because known t-elements are located close to and in the same transcriptional orientation as active genes (18S-5S, 26S, tRNA(Pro)) in wheat mtDNA, our results raise the question of whether t-elements play a role in gene expression in wheat mitochondria.