A yeast mutant which accumulates precursor tRNAs.

It has been proposed that the conditional yeast mutant ts136 is defective in the transport of mRNA from the nucleus to the cytoplasm (Hutchinson, Hartwell and McLaughlin, 1969). We have examined ts136 to determine whether it is defective in tRNA biosynthesis. At the restrictive temperature, the mutant accumulates twelve new species of RNA. These species co-migrate on polyacrylamide gels with some of the pulse-labeled precursor tRNAs. Three of the new RNAs (species 1a, 1b and 1c are large enough to contain two tandom tRNAs. Although RNAs 1a, 1b, and 1c do not contain detectable levels of modified and methylated bases, at least one of them hybridizes to DNA from an E. coli plasmid containing a yeast tRNA gene. All the remaining RNAs (2--8) contain modified and methylated bases typical of tRNA. Three of these species were tested and were found to hybridize to tRNA genes. Ribosomal RNA synthesis is also defective in ts136. It is suggested that ts136 may be defective in a nucleolytic activity, which is a prerequisite to RNA transport.     

Identity elements required for enzymatic formation of N2,N2-dimethylguanosine from N2-monomethylated derivative and its possible role in avoiding alternative conformations in archaeal tRNA

Here, we have investigated the specificity of purified recombinant tRNA:m(2)(2)G10 methyltransferase of Pyrococcus abyssi ((Pab)Trm-m(2)(2)G10 enzyme). This archaeal enzyme catalyses mono- and dimethylation of the N(2)-exocyclic amino group of guanine at position 10 of several tRNA species. Our results indicate that only few identity elements are required for the efficient formation of m(2)(2)G10. They are composed of a G10.U25 wobble base-pair in the dihydrouridine arm (D-arm) and a four nucleotide variable loop (V-loop) within a canonical three-dimensional (3D) structure. The types of base-pairs in the D-arm or amino acid acceptor stem are also important for the enzymatic reaction, but appear to affect only the rate of tRNA methylation. However, in tRNA species harbouring a G10-C25 Watson-Crick base-pair and/or five nucleotide V-loop, only m(2)G10 is produced. To impair the monomethylation reaction, drastic amputation in the T-arm is required. Our observations contrast with those reported earlier for the identity elements required for a remotely related Pyrococcus furiosus Trm-m(2)(2)G26 enzyme (alias (Pfu)Trm1) that also catalyses the two step formation of m(2)(2)G but at position 26 in several tRNA species. In this case, a G10-C25 base-pair together with the five nucleotide V-loop were shown to be required for efficient formation of m(2)(2)G26. Thus, in the Pyrococcus genus, the major identity elements that preclude formation of m(2)(2)G at positions 10 or 26 in tRNA are mutually exclusive. Therefore, the Trm-m(2)(2)G10 and Trm-m(2)(2)G26 enzymes have evolved independently towards different specificities. In addition, identity elements for m(2)/m(2)(2)G10 formation in archaeal tRNA are different from the ones required for m(2)G10 formation in eukaryal tRNA. We propose that archaeal tRNA:m(2)(2)G10 methyltransferases, unlike the orthologous eukaryal tRNA:m(2)G10 methyltransferases, evolved towards m(2)(2)G10 specificity due to the possible requirement of preventing formation of alternative structures in G/C rich archaeal tRNA species.     

The specificities of four yeast dihydrouridine synthases for cytoplasmic tRNAs

Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridine and dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U(16) and U(17), Dus2p for U(20), Dus3p for U(47), and Dus4p for U(20a) and U(20b).     

A neutron investigation of yeast valyl-tRNA synthetase interaction with tRNAs.

A new way of studying RNA-protein complexes, using neutron small angle scattering in solution, is described and was applied in the case of the system, yeast valyl-tRNA synthetase, interacting with its cognate and non cognate yeast tRNAs. It was shown that, when limited amounts of tRNA (either cognate or non cognate) are added to valyl-tRNA synthetase, a complex consisting of two enzyme molecules and one tRNA molecule is first formed. It is subsequently dissociated to a one to one complex when more tRNA is present in the solution. The association curve shows a maximum for a molecular ratio, enzyme over tRNA, equal to 2.     

Study of yeast mitochondrial tRNAs by two-dimensional polyacrylamide gel electrophoresis: characterization of isoaccepting species and search for imported cytoplasmic tRNAs.

By two-dimensional polyacrylamide gel electrophoresis, yeast mitochondrial tRNA is fractionated into 27 major species. All but 6 of them migrate distinctly from cytoplasmic tRNAs. Migration of mitochondrial DNA-coded mitochondrial tRNAs shows the occurence of only one cytoplasmic tRNA in mitochondria. Several mitochondrial tRNA spots are identified on the electrophoregrams, some of them show isoaccepting species (Val, Ser, Met, Leu). It is suggested that there are sufficient mitochondrial tRNA genes on yeast mitochondrial DNA to allow mitochondrial protein biosynthesis by the mitochondrial tRNAs alone. Guanosine + Cytidine content and rate base composition are reported for some individual species. Mitochondrial tRNAPhe lacks Ribothymidine.     

Comparison of dissimilarity patterns of E coli, yeast and mammalian tRNAs.

A number of experimental approaches have been developed for identification of recognition (identity) sites in tRNAs. Along with them a theoretical methodology has been proposed by McClain et al that is based on concomitant analysis of all tRNA sequences from a given species. This approach allows an evaluation of nucleotide combinations present in isoacceptor tRNAs specific for the given amino acid, and not present in equivalent positions in cloverleaf structure in other tRNAs of the same organism. These elements predicted from computer analysis of the databank could be tested experimentally for their participation in forming recognition sites. The correlation between theoretical predictions and experimental data appeared promising. The aim of the present work consisted of introducing further improvements into McClain's procedure by: i), introducing into analysis a variable region in tRNAs which had not been previously considered; to accomplish this, 'normalization' of variable nucleotides was suggested, based on primary and tertiary structures of tRNAs; ii), developing a new procedure for comparison of patterns for synonymous and non-synonymous tRNAs from different organisms; iii), analysis of 3- and 4-positional contacts between tRNAs and enzymes in addition to a formerly used 2-positional model. A systematic application of McClain's procedure to mammalian, yeast and E coli tRNAs led to the following results: i), imitancy patterns for non-synonymous tRNAs of any amino acid specificity and from any organisms analysed so far overlap by no more than 30%, providing a structural basis for discrimination with high fidelity between cognate and non-cognate tRNAs; ii), the predicted identity sites are non-randomly distributed within tRNA molecules; the dominant role is ascribed to only two regions--anticodon and amino acid stem which are located far apart from one another at extremes of all tRNA molecules; iii), the imitancy patterns for synonymous tRNAs in lower (yeast) and higher (mammalian) eukaryotes are similar but not identical; iv), distribution of predicted identity sites in the cloverleaf structure in prokaryotes and eukaryotes is essentially different: in eubacterial tRNAs the major role in recognition plays anticodon and/or amino acid acceptor stem, whereas in eukaryotic (both unicellular and multicellular) tRNAs the remaining part of the molecules is also involved in recognition; v), the imitancy patterns of synonymous tRNAs from prokaryotes and eukaryotes are dissimilar, this observation leads to the prediction that the tRNA identity sites for the same amino acid in prokaryotes and eukaryotes may differ.     

Identity elements and aminoacylation of plant tRNATrp.

Mutation of the Arabidopsis thaliana tRNA (Trp)(CCA) anticodon or of the A73 discriminator base greatly diminishes in vitro aminoacylation with tryptophan, indicating the importance of these nucleotides for recognition by the plant tryptophanyl-tRNA synthetase. Mutation of the tRNA (Trp)(CCA) anticodon to CUA so as to translate amber nonsense codons permits tRNA (Trp)(CCA) to be aminoacylated by A.thaliana lysyl-tRNA synthetase. Thus, translational suppression by tRNA (TRP)(CCA) observed in plant cells includes significant incorporation of lysine into protein.     

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.     

Sequence analysis of ARS elements in fission yeast.

Chromosomal DNA of Schizosaccharomyces pombe contains sequences with properties analogous to ARS elements of Saccharomyces cerevisiae. Following Sau3A fragmentation of the S. pombe genome we have recovered a number of such fragments in an M13-based shuttle vector, suitable for subsequent sequence analysis. The complete nucleotide sequence has been obtained for eight ARS+ inserts derived from the Sau3A cloning and for the ARS present in pFL20 isolated previously by Losson and Lacroute (Cell, 32, 371-377, 1983). The Sau3A clones are single fragments between 0.8 and 1.8 kb. No ARS+ clones smaller than this were recovered even though the average size Sau3A fragment in S. pombe is approximately 200-300 bp. The sequence analysis revealed that all clones are AT-rich (69-75% A + T residues), and all contain a particularly AT-rich 11 bp core element represented by the consensus sequence 5' (A/T)PuTT-TATTTA(A/T) 3'. Deletion mapping indicates that the consensus in all cases is in the vicinity of a functional ARS domain. However precise excision of the consensus by in vitro mutagenesis has little effect on ARS activity as judged by the transformation assay. We argue that the association of the consensus with the ARS domain occurs too reproducibly to be explained by chance alone. We suggest that although it may not be essential for the extrachromosomal maintenance of plasmids in S. pombe, the consensus does have a function in situ in the chromosome and thus is always present as a cryptic sequence in the isolated ARS element.     

Primary structure of three tRNAs from brewer's yeast: tRNAPro2, tRNAHis1 and tRNAHis2

The primary structures of three brewer's yeast tRNAs: tRNAPro2 and tRNAHis1 and 2 have been determined (Formula:see text) The U* in the anticodon U*-G-G of tRNAPro2 is probably a derivative of U; tRNAPro2 has 80 per cent homology with mammalian tRNAsPro. tRNAHis1 and tRNAHis2 differ by only 5 nucleotides; they have identical anticodons and may therefore recognize both codons for histidine; they have an additional nucleotide at the 5' end. As in all other sequenced tRNAsHis this nucleotide is not paired with the fourth nucleotide from acceptor adenosine. All three sequenced tRNAs have a low degree of homology with their counterparts from yeast mitochondria.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

The N¹-methyl-Adenosine (m¹A58) modification at the conserved nucleotide 58 in the TΨC loop is present in most eukaryotic tRNAs. In yeast, m¹A58 modification is essential for viability because it is required for the stability of the initiator-tRNA^Met. However, m¹A58 modification is not required for the stability of several other tRNAs in yeast. This differential m¹A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m¹A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m¹A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA^Met is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m¹A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m¹A58 hypomodified tRNAs. Most m¹A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m¹A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.     

Two isoaccepting seryl tRNAs coded by separate mitochondrial genes in yeast

Summary In S. cerevisiae four isoacceptor mitochondrial tRNAs for serine have been separated by reversed phase chromatography. At least two of these species are products of different genes. In this work the deletion mapping technique has been used to locate two genes for tRNA^ser. The gene for tRNA^ser previously localized in the oli I region of the mitochondrial genome has been found to code for tRNA _ser ² , and another gene coding for tRNA _ser ¹ has been detected in the region where most of other tRNA genes are found. Results of fine mapping experiments allowed to localize this gene in the proximity of the gene for tRNA^arg.     

Influence of various factors on the recognition specificity of tRNAs by yeast valyl-tRNA synthetase.

Using filtration through nitrocellulose membranes we found that complexes between yeast valyl-tRNA synthetase can easily be detected at low pH and ionic strength with the cognate tRNAVal, but also with several non-cognate tRNAs (tRNAPhe, tRNATyr, tRNAMet and tRNAAsp). We show here that the amino acid linked to the tRNA has no detectable effect on these interactions. The influence of various factors on the discrimination by the enzyme between the cognate and the non-cognate tRNAs has been studied. An increase in pH or ionic strength leads to a decrease in the same ratio of the affinity constants between the enzyme and the cognate as well as the noncognate tRNA. The addition of organic solvents has little effect on these constant either in the cognate or in the non-cognate systems; the addition of substrates of the aminoacylation reaction has not effect on the ratio between the constants. This similar behaviour suggests that at least part of the specific of non-specific interactions must be identical. On the contrary, magnesium between 1 mM and 50 mM increases the specificity of recognition, showing the importance of slight conformational changes in the tRNA molecule to the specificity of interaction.     

Functional conservation of multiple elements in yeast chromosomal replicators.

Replicators that control the initiation of DNA replication in the chromosomes of Saccharomyces cerevisiae retain their function when cloned into plasmids, where they are commonly referred to as autonomously replicating sequences (ARSs). Previous studies of the structure of ARS1 in both plasmid and chromosome contexts have shown that it contains one essential DNA element, A, that includes a match to the ARS consensus sequence (ACS), and three additional elements, B1, B2, and B3, that are also important for ARS function. Elements A and B3 are bound by a candidate initiator protein called the origin recognition complex and ARS-binding factor 1, respectively. Although the A and B3 elements have been found in other ARSs, sequence comparisons among ARSs have failed to identify B1- and B2-like elements. To assess the generality of the modular nature of yeast replicators, linker substitution mutagenesis of another yeast chromosomal replicator, ARS307, was performed. Three DNA sequence elements were identified in ARS307, and they were demonstrated to be functionally equivalent to the A, B1, and B2 elements present in ARS1. Despite the lack of DNA sequence similarity, the B1 and B2 elements at each ARS were functionally conserved. Single-base substitutions in the core of the ARS1 B1 and B2 elements identified critical nucleotides required for the function of the B1 element. In contrast, no single-point mutations were found to affect B2 function. The results suggest that multiple DNA sequence elements might be a general and conserved feature of replicator sequences in S. cerevisiae.