The nucleotide sequence of the major glutamate transfer RNA from Schizosaccharomyces pombe.

The nucleotide sequence of glutamate tRNA1 from Schizosaccharomyces pombe was determined to be pU-C-C-G-U-U-G-U-m1G-G-U-C-C-A-A-C-G-G-C-D-A-G-G-A-U-U-C-G-U-C-G-C-U-U-U*-C-A-C-C-G-A-C-G-G-G-A-G-m5C-G-G-G-G-T-psi-C-G-A-C-U-C-C-C-C-G-C-A-A-C-G-G-A-G-C-C-AOH. The sequence differs markedly from that of S. cerevisiae tRNAGlu. S. pombe glutamate tRNA1 can be aminoacylated by the homologous glutaminyl-tRNA synthetase as well as by the corresponding enzyme from S. cerevisiae.     

Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase.     

Aminoacylation of anticodon loop substituted yeast tyrosine transfer RNA

A procedure for replacing residues 33-35 in the anticodon loop of yeast tRNATyr with any desired oligonucleotide has been developed. The three residues were removed by partial ribonuclease A digestion. An oligonucleotide was inserted into the gap in four steps by using RNA ligase, polynucleotide kinase, and pseT 1 polynucleotide kinase. The rate of aminoacylation of anticodon loop substituted tRNATyr by yeast tyrosyl-tRNA synthetase was found to depend upon the sequence of the oligonucleotide inserted. This suggests that the nucleotides in the anticodon loop of yeast tRNATyr are required for optimal aminoacylation. In addition, tRNATyr modified to have a phenylalanine anticodon was shown to be misacylated by yeast phenylalanyl-tRNA synthetase at a rate at least 10 times faster than unmodified tRNATyr. Thus, the anticodon is used by phenylalanyl-tRNA synthetase to distinguish between tRNAs.     

Cloning and sequence determination of the gene encoding the largest subunit of the fission yeast Schizosaccharomyces pombe RNA polymerase I

The gene encoding the largest subunit of RNA polymerase I (SPRPA190) was cloned from the fission yeast Schizosaccharomyces pombe by cross-hybridization with a probe containing part of the corresponding Saccharomyces cerevisiae gene RPA190. The SPRPA190 gene is present in a single copy per haploid genome and is essential for cell growth. The polypeptide encoded by this gene, as deduced from the nucleotide sequence of the uninterrupted coding frame, consists of 1689 amino acids and its calculated Mr is 189,300. The amino acid identity between the subunits of the two yeast species is 50%. Amino acid sequence conservation covers the regions previously suggested to be functionally important for the S. cerevisiae enzyme. In addition, two markedly hydrophilic regions recognized in the S. cerevisiae polypeptide can also be recognized in the S. pombe polypeptide in approximately the same positions, even though the amino acid sequences in these regions are diverged from each other. In the 5'-flanking region of the gene, several nucleotide sequence elements are detected which are also found in the two S. pombe ribosomal protein genes so far sequenced.     

Specific interaction of anticodon loop residues with yeast phenylalanyl-tRNA synthetase

Thirteen different yeast tRNAPhe variants with single nucleotide changes in positions 34-37 in the anticodon region were prepared by an enzymatic procedure described previously. Aminoacylation kinetics using purified yeast phenylalanyl-tRNA synthetase revealed that the level of aminoacylation was very different for different sequences inserted. The low level of aminoacylation was the result of a steady state between a slow forward reaction rate and spontaneous deacylation of the product. Aminoacylation kinetics performed at higher synthetase concentrations revealed that substitution at position 34 in tRNAPhe decreased the Km nearly 10-fold but only had a small effect on Vmax. Similar substitutions at positions 35, 36, and 37 had a lesser effect. These data suggest a sequence-specific contact between the anticodon of yeast tRNAPhe and the cognate synthetase.     

Mechanism of discrimination between cognate and non-cognate tRNAs by phenylalanyl-tRNA synthetase from yeast.

The interaction between phenylalanyl-tRNA synthetase from yeast and Escherichia coli and tRNAPhe (yeast), tRNASer (yeast), tRNA1Val (E. coli) has been investigated by ultracentrifugation analysis, fluorescence titrations and fast kinetic techniques. The fluorescence of the Y-base of tRNAPhe and the intrinsic fluorescence of the synthetases have been used as optical indicators. 1. Specific complexes between phenylalanyl-tRNA synthetase and tRNAPhe from yeast are formed in a two-step mechanism: a nearly diffusion-controlled recombination is followed by a fast conformational transition. Binding constants, rate constants and changes in the quantum yield of the Y-base fluorescence upon binding are given under a variety of conditions with respect to pH, added salt, concentration of Mg2+ ions and temperature. 2. Heterologous complexes between phenylalanyl-tRNA synthetase (E. coli) and tRNAPhe (yeast) are formed in a similar two-step mechanism as the specific complexes; the conformational transition, however, is slower by a factor 4-5. 3. Formation of non-specific complexes between phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) proceeds in a one-step mechanism. Phenylalanyl-tRNA synthetase (yeast) binds either two molecules of tRNAPhe (yeast) or only one molecule of tRNATyr (E. coli); tRNA1Val (E. coli) or tRNASer (yeast) are also bound in a 1:1 stoichiometry. Binding constants for complexes of phenylalanyl-tRNA synthetase (yeast) and tRNATyr (E. coli) are determined under a variety of conditions. In contrast to specific complex formation, non-specific binding is disfavoured by the presence of Mg2+ ions, and is not affected by pH and the presence of pyrophosphate. The difference in the stabilities of specific and non-specific complexes can be varied by a factor of 2--100 depending on the ionic conditions. Discrimination of cognate and non-cognate tRNA by phenylalanyl-tRNA synthetase (yeast) is discussed in terms of the binding mechanism, the topology of the binding sites, the nature of interacting forces and the relation between specificity and ionic conditions.     

Regulation of E.coli phenylalanyl-tRNA synthetase operon in vivo

The phenylalanyl-tRNA synthetase operon is composed of two adjacent, cotranscribed genes, pheS and pheT, corresponding respectively to the small and large subunit of phenylalanyl-tRNA synthetase. A fusion between the regulatory regions of phenylalanyl-tRNA synthetase operon and the lac structural genes has been constructed to study the regulation of the operon. The pheS,T operon was shown, using the fusion, to be derepressed when phenylalanine concentrations were limiting in a leaky auxotroph mutated in the phenylalanine biosynthetic pathway. Furthermore, a mutational alteration in the phenylalanyl-tRNA synthetase gene, bradytrophic for phenylalanine, was also found to be derepressed under phenylalanine starvation. These results indicate that the pheS,T operon is derepressed when the level of tRNAPhe aminoacylation is lowered. By analogy with other well-studied amino acid biosynthetic operons known to be controlled by attenuation, these in vivo results indicate that phenylalanyl-tRNA synthetase levels are controlled by an attenuation-like mechanism.     

The nucleotide sequence of phenylalanine tRNA and 5S RNA from Rhodospirillum rubrum

The complete nucleotide sequence of tRNAPhe and 5S RNA from the photosynthetic bacterium Rhodospirillum rubrum has been elucidated. A combination of in vitro and in vivo labelling techniques was used. The tRNAPhe sequence is 76 nucleotides long, 7 of which are modified. The primary structure is typically prokaryotic and is most similar to the tRNAPhe of Escherichia coli and Anacystis nidulans (14 differences of 76 positions). The 5S ribosomal RNA sequence is 120 nucleotides long and again typical of other prokaryotic 5S RNAs. The invariable GAAC sequence is found starting at position 45. When aligned with other prokaryotic 5S RNA sequences, a surprising amount of nucleotide substitution is noted in the prokaryotic loop region of the R. rubrum 5S RNA. However, nucleotide complementarity is maintained reinforcing the hypothesis that this loop is an important aspect of prokaryotic 5S RNA secondary structure. The 5S and tRNAPhe are the first complete RNA sequences available from the photosynthetic bacteria.     

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.     

The nucleotide sequence of 5S ribosomal RNA from Schizosaccharomyces pombe

The nucleotide sequence of 5S rRNA from the fission yeast, S. pombe, has been established by post labeling procedures combined with cataloging RNase T1- and A-oligonucleotides derived from unlabeled 5S rRNA. The sequence consists of 119 nucleotides without a modified base and shows more dissimilarities (at 38 positions) from that of S. cerevisiae than from that of humans (at 33 positions).     

Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation.

Squalene synthetase (farnesyl diphosphate:farnesyl diphosphate farnesyltransferase; EC 2.5.1.21) is thought to represent a major control point of isoprene and sterol biosynthesis in eukaryotes. We demonstrate structural and functional conservation between the enzymes from humans, a budding yeast (Saccharomyces cerevisiae), and a fission yeast (Schizosaccharomyces pombe). The amino acid sequences of the human and S. pombe proteins deduced from cloned cDNAs were compared to those of the known S. cerevisiae protein. All are predicted to encode C-terminal membrane-spanning proteins of approximately 50 kDa with similar hydropathy profiles. Extensive sequence conservation exists in regions of the enzyme proposed to interact with its prenyl substrates (i.e., two farnesyl diphosphate molecules). Many of the highly conserved regions are also present in phytoene and prephytoene diphosphate synthetases, enzymes which catalyze prenyl substrate condensation reactions analogous to that of squalene synthetase. Expression of cDNA clones encoding S. pombe or hybrid human-S. cerevisiae squalene synthetases reversed the ergosterol requirement of S. cerevisiae cells bearing ERG9 gene disruptions, showing that these enzymes can functionally replace the S. cerevisiae enzyme. Inhibition of sterol synthesis in S. cerevisiae and S. pombe cells or in cultured human fibroblasts by treatment with the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor lovastatin resulted in elevated levels of squalene synthetase mRNA in all three cell types.     

Effect of excision of the Y-base on the interaction of tRNAPhe (yeast) with phenylalanyl-tRNA synthetase (yeast).

The interaction between tRNAPhe (yeast), from which the Y-base has been removed by acid treatment, and phenylalanyl-tRNA synthetase (yeast) has been investigated by fluorescence competition titrations and sedimentation velocity runs. The binding parameters are given under various ionic conditions. The tRNAPhe-Y still can occupy the specific binding sites on the enzyme. Compared to unmodified tRNAPhe, the binding constant is lowered by more than one order of magnitude. It can be concluded that the Y-base is not necessary for specific recognition of tRNAPhe by the cognate synthetase, it rather may represent a point of attachment for the synthetase.     

The 5.8S RNA gene sequence and the ribosomal repeat of Schizosaccharomyces pombe

We have characterized the rRNA gene repeat in Schizosaccharomyces pombe. This repeat, which does not contain the 5S RNA gene, is found in a 10.4 kb HindIII DNA fragment. We have determined the nucleotide sequences of the S. pombe 5.8S RNA gene and intergenic spacers from two different 10.4 kb DNA fragments. Analysis of isolated total cellular 5.8S RNA revealed the presence of eight species of 5.8S RNA, differing in the number of nucleotides at the 5'-end. The eight 4.8S RNA species vary in length from 158 to 165 nucleotides. Apart from the heterogeneity observed at the 5'-end, the sequence of the eight 5.8S RNA species appears to be identical and is the same sequence as coded for by the 5.8S genes. The gene sequence shows great homology to the 5.8S RNA genes or S. cerevisiae and N. crassa. Most of the base differences are confined to the highly variable stem though to be involved in co-axial helix stacking with the 25S RNA, where base pairing is nearly identical despite the sequence differences. Secondary structure models are examined in light of 5.8S RNA oligonucleotide conservation across species from yeasts to higher eukaryotes.     

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.     

Incorporation of 1,N6-ethenoadenosine into the 3' terminus of tRNA using T4 RNA ligase. 1. Preparation of yeast tRNAPhe derivatives

The 3'-terminal A-C-C-A sequence of yeast tRNAPhe has been modified by replacing either adenosine 76 or 73 with the fluorescent analogues 1,N6-ethenoadenosine (epsilon A) or 2-aza-1,N6-ethenoadenosine (aza-epsilon A). T4 RNA ligase was used to join the nucleoside 3',5'-bisphosphates to the 3' end of the tRNA which was shortened by one [tRNAPhe(-A)] or four [tRNAPhe(-ACCA)] nucleotides. It was found that the base-paired 3'-terminal cytidine 72 in tRNAPhe(-ACCA) is a more efficient acceptor in the ligation reaction than the unpaired cytidine 75 at the A-C-C terminus of tRNAPhe(-A). This finding indicates that the mobility of the accepting nucleoside substantially influences the ligation reaction, the efficiency being higher the lower the mobility. This conclusion is corroborated by the observation that the ligation reaction with the double-stranded substrate exhibits a positive temperature dependence rather than a negative one as found for single-stranded acceptors. The replacement of the 3'-terminal adenosine 76 with epsilon A and aza-epsilon A leads to moderately fluorescent tRNAPhe derivatives, which are inactive in the aminoacylation reaction. A number of other tRNAs (Met, Ser, Glu, Lys and Leu-specific tRNAs both from yeast and Escherichia coli) are also inactivated by epsilon A incorporation. Replacement of adenosine 73 followed by repair of the C-C-A end using nucleotidyl transferase leads to tRNAPhe derivatives which are fully active in the aminoacylation reaction and in polyphenylalanine synthesis. The fluorescence of epsilon A and aza-epsilon A at position 73 is virtually completely quenched, suggesting a stacked arrangement of bases around this position. There is no fluorescence increase when the epsilon A-labeled tRNAPhe is complexed with phenylalanyl-tRNA synthetase, elongation factor Tu, or ribosomes. These observations indicate that the stacked conformation of the 3' terminus is not changed appreciably in these complexes.     

Functional mutants of phenylalanine transfer RNA from Escherichia coli.

The gene pheV from Escherichia coli, coding for tRNAPhe and carried on a plasmid, has been mutagenised with hydroxylamine. Mutants in the structural gene have been identified using two criteria: (i) de-attenuation of beta-galactosidase expression, while under the control of the attenuator region of the pheS,T operon by means of an operon fusion; (ii) loss of ability to complement thermosensitivity of a mutant Phe-tRNA synthetase. Mutants showing de-attenuation were sequenced and two nucleotide changes identified: G44----A44 (found five times) and m7G46----A46 (found once). Sequencing of mutants that lost complementation identified two further tRNA mutants, C2---U2 and G15----A15; the mutant m7G46----A46 was also re-isolated by this criterion. Three of the mutants involve bases implicated in tertiary rather than secondary structure hydrogen bonding. One hypothesis for the mechanism of de-attenuation is that mutant tRNAPhe molecules compete with the wild-type tRNAPhe on the ribosome but are inefficient at some step in the elongation process.     

The Rpb4 Subunit of Fission Yeast Schizosaccharomyces pombe RNA Polymerase II Is Essential for Cell Viability and Similar in Structure to the Corresponding Subunits of Higher Eukaryotes

Both the gene and the cDNA encoding the Rpb4 subunit of RNA polymerase II were cloned from the fission yeast Schizosaccharomyces pombe. The cDNA sequence indicates that Rpb4 consists of 135 amino acid residues with a molecular weight of 15,362. As in the case of the corresponding subunits from higher eukaryotes such as humans and the plant Arabidopsis thaliana, Rpb4 is smaller than RPB4 from the budding yeast Saccharomyces cerevisiae and lacks several segments, which are present in the S. cerevisiae RPB4 subunit, including the highly charged sequence in the central portion. The RPB4 subunit of S. cerevisiae is not essential for normal cell growth but is required for cell viability under stress conditions. In contrast, S. pombe Rpb4 was found to be essential even under normal growth conditions. The fraction of RNA polymerase II containing RPB4 in exponentially growing cells of S. cerevisiae is about 20%, but S. pombe RNA polymerase II contains the stoichiometric amount of Rpb4 even at the exponential growth phase. In contrast to the RPB4 homologues from higher eukaryotes, however, S. pombe Rpb4 formed stable hybrid heterodimers with S. cerevisiae RPB7, suggesting that S. pombe Rpb4 is similar, in its structure and essential role in cell viability, to the corresponding subunits from higher eukaryotes. However, S. pombe Rpb4 is closer in certain molecular functions to S. cerevisiae RPB4 than the eukaryotic RPB4 homologues.