Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.     

Coupling of protein synthesis and mitochondrial import in a homologous yeast in vitro system.

We made use of a homologous cell-free mitochondrial protein import system derived from the yeast Saccharomyces cerevisiae to investigate the coupling of protein synthesis and import. Mitochondrial precursor proteins were synthesized in a yeast lysate either in the presence or absence of isolated yeast mitochondria. We were, therefore, able to analyze protein import into mitochondria either in a strictly posttranslational reaction (when isolated mitochondria were added only after protein synthesis has been arrested by the addition of cycloheximide) or in a reaction in which synthesis and import were permitted to occur simultaneously. We found that the import of a precursor protein consisting of the amino-terminal mitochondrial targeting sequence of cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase is very inefficient in a strictly posttranslational reaction, whereas efficient import is observed if precursor synthesis and import are coupled. The same result was obtained when we analyzed the import of bulk endogenous yeast mitochondrial proteins in this system. Finally, we found that the insertion of the yeast outer membrane protein porin is also several times more efficient when synthesis and insertion are coupled.     

Protein import into mitochondria in a homologous yeast in vitro system

To study the import of proteins into mitochondria we developed a homologous in vitro system in which mitochondria and cell-free translation extract are both derived from the yeast Saccharomyces cerevisiae. This system allows the synthesis of precursor proteins in the presence of isolated mitochondria and offers a means of analyzing yeast mutants defective in mitochondrial protein import. The in vitro import of an artificial precursor protein into yeast mitochondria in the presence of its substrate analog was analyzed subsequent to synthesis in either a yeast or rabbit reticulocyte cell-free translation reaction. Results suggest that a component(s) present in the yeast cytosolic extract may interact with the precursor protein.     

First position wobble in codon-anticodon pairing: amber suppression by a yeast glutamine tRNA

A 2.4-kb fragment of DNA isolated from the Saccharomyces cerevisiae genome was found to suppress amber mutations when its carrier plasmid was present in high copy number. A 1.2-kb subclone of this fragment was sufficient to confer suppressor activity. Sequencing has established that this fragment carries a normal glutamine tRNA gene. Deletion of this tRNA gene from the subclone resulted in the loss of suppressor activity. The tRNAGln has the anticodon CUG that normally recognizes the glutamine codon CAG. We propose that suppression occurs via an inefficient readthrough of the UAG amber stop codons during translation. Such readthrough requires wobble in the first position of the codon.     

Nucleotide sequence of a yeast tRNAArg3A gene and its transcription in a homologous in vitro system

Neutrophil homogenates contained a high affinity guanosine triphosphatase (GTPase) that was stimulatable (+27%) by the addition of 100 nM N-formyl chemotactic peptide (CHO-pep), but not by 1 microgram X ml-1 phorbolmyristate acetate (PMA). Kinetic analysis of the stimulation demonstrated an apparent lagtime of 14.3 +/- 6.9 s between the addition of CHO-pep and the optimal GTPase stimulation. The GTPase activity (but not CHO-pep-stimulated GTPase activity) was preserved in a highly purified plasma membrane fraction of the homogenate. From these observations we suggest that both a high affinity guanine nucleotide binding protein and GTPase are closely associated with the plasma membrane CHO-pep receptor. The possibility that GTPase activity may influence guanine nucleotide regulation of adenylate cyclase during CHO-pep stimulation of neutrophils is discussed.     

Pairing properties of the methylester of 5-carboxymethyl uridine in the wobble position of yeast tRNA3Arg.

At optimum magnesium concentration (10 mM) both yeast tRNA1Arg and tRNA3Arg are able to bind to poly (A,G) and A-G-A in presence of Escherichia coli robisomes. With A-G-G only tRNA1Arg ginds, wherea tRNA3Arg (anticodon mcm5 U-C-U) is not bound. This result means that the methylcarboxymethyl substituant in position 5 of U prevents its wobble with G.     

Presence of the methylester of 5-carboxymethyl uridine in the wobble position of the anticodon of tRNAIII Arg from brewer's yeast.

The methylester of 5-carboxymethyluridine (mcm5U), its degradation product 5-carboxymethyluridine (cm5U) and the corresponding nucleotide (cm5Up) were isolated from brewer's yeast tRNAIII Arg or from the dodecanucleotide containing the anticodon. Their chromatographic and electrophoretic properties and their UV absorbing spectra were identical to that of the corresponding synthetic compounds. The gas chromatographic behavior and the mass spectrum of mcm5U obtained from tRNAIII Arg and of a synthetic sample were also identical ; the rare occurence of a thermal reciprocal bimolecular methyl-hydrogen transfer in the mass spectrometer ion source was observed. A mild alkaline treatment of tRNAIII Arg leads to the saponification of mcm5U into cm5U (within the tRNA), which can be again esterified in the presence of a yeast homogenate and (methyl-14C) S adenosylmethionine. The radioactivity was found in the mcm5U located in the wobble position of the anticodon of tRNAIII Arg. The presence of this odd nucleotide in that position could possibly restrict the codon-anticodon interaction of tRNAIII Arg.     

The kinetics of binding of U-U-C-A to a dodecanucleotide anticodon fragment from yeast tRNA-Phe.

The kinetics of U-U-C-A binding to the dodecanucleotide (A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-Gp) isolated from the anticodon region of yeast tRNA-Phe are similar to the kinetics of binding of U-U-C-A to intact tRNA-Phe. A large enhancement in binding constant over that predicted for U-U-C-A-U-G-A-A is observed for both the complexes of dodecanucleotide and tRNA-Phe with U-U-C-A. This strongly suggests that both the anticodon loop in tRNA-Phe and the dodecanucleotide can form four base pairs with U-U-C-A. Furthermore, the enhanced stability cannot be attributed to a special conformation of the anticodon loop, but instead the anticodon loop is probably flexible. A likely explanation for the increased binding is the effect of non-base-paired ends. This increased thermodynamic stability comes from a larger entropy gain rather than a larger enthalpy decrease.     

A multicomponent protein of a fission yeast that promotes joint molecule formation from homologous DNAs.

We developed a quantitative assay ("homologous pairing gel assay") adequate for the purification of the activity promoting the formation of joint molecules, an intermediate of homologous recombination ("homologous pairing"). With this assay, one can measure the extent of homologous pairing between a single-stranded DNA and a strand of 3H-labeled double-stranded DNA by crude enzyme preparations. Since the total activity did not significantly change during the sporulation process, we tried to purify the activity from a whole cell extract of mitotic cells of a fission yeast, (Schizosaccharomyces pombe). Through quantitative assaying of a single fraction or of mixed fractions, we obtained three fractions, all of which were required for the maximum level of the ATP-independent homologous pairing: Fractions 65, 100, and 30. In Fractions 100 and 30, polypeptides of approximately 100 and approximately 30 kDa (the 100- and 30-kDa polypeptides), respectively, were the sole detectable components. Fraction 65 contained a polypeptide of approximately 65 kDa (the 65-kDa polypeptide) as the major component and also small amounts of the 30- and 100-kDa polypeptides. Fraction 65 by itself promoted homologous pairing, but the reaction was saturated at a level of approximately 20% of the maximum level achieved with the recA protein. Even when added in excess, Fraction 30 or 100 alone did not promote detectable homologous pairing. A mixture of Fractions 65 and 100 at a rather strict optimum ratio only promoted homologous pairing, the level being 50-70% that with the recA protein, suggesting a stoichiometric complex of these polypeptides as the active form. Fraction 30 alone did not enhance the reaction with Fraction 65, but stimulated homologous pairing promoted by the optimum mixture of Fractions 65 and 100 to the maximum level achieved with the recA protein. Therefore, the homologous pairing-promoting protein from the fission yeast is likely to be a multicomponent protein.     

Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.     

Participation of yeast inosine 5'-monophosphate dehydrogenase in an in vitro complex with a fragment of the C-rich telomeric strand

As part of our investigation of the i-motif, an intercalated structure formed by C-rich nucleic acid sequences, we searched for proteins of Saccharomyces cerevisiae which could associate with a sequence of the C-rich telomeric strand, d((CCCACA)(3)CCC). A gel retardation assay of yeast protein extract, in conditions where the DNA fragment folds into an intramolecular i-motif, shows formation of one major retarded band. The retarding factor was further characterized by a differential affinity procedure using streptavidin beads coated (or not coated) with the biotin-labeled DNA fragment. Differentially bound proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by mass spectroscopy and Edman degradation as Imd2p, Imd3p and Imd4p. These highly similar (>95%) proteins are analogs of the two human NAD-dependent inosine 5'-monophosphate dehydrogenases (IMPDH) which occur as tetramers. The mass of the protein, as determined by gel exclusion chromatography, is about 250 kDa and is compatible with an IMPDH tetramer, but other compositions, involving non-IMPDH components, are not excluded. We note that the genes coding for Imd2p and Imd3p are located close to the telomere, and could therefore be subject to silencing by the telomere position effect.     

Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation

The cell wall of fungal cells is important for cell integrity and cell morphogenesis and protects against harmful environmental conditions. The yeast cell wall is a complex structure consisting mainly of mannoproteins, glucan, and chitin. The molecular mechanisms by which the cell wall components are synthesized and transported to the cell surface are poorly understood. We have identified and characterized two homologous yeast proteins, Sbe2p and Sbe22p, through their suppression of a chs5 spa2 mutant strain defective in chitin synthesis and cell morphogenesis. Although sbe2 and sbe22 null mutants are viable, sbe2 sbe22 cells display several phenotypes indicative of defects in cell integrity and cell wall structure. First, sbe2 sbe22 cells display a sorbitol-remediable lysis defect at 37 degrees C and are hypersensitive to SDS and calcofluor. Second, electron microscopic analysis reveals that sbe2 sbe22 cells have an aberrant cell wall structure with a reduced mannoprotein layer. Finally, immunofluorescence experiments reveal that in small-budded cells, sbe2 sbe22 mutants mislocalize Chs3p, a protein involved in chitin synthesis. In addition, sbe2 sbe22 diploids have a bud-site selection defect, displaying a random budding pattern. A Sbe2p-GFP fusion protein localizes to cytoplasmic patches, and Sbe2p cofractionates with Golgi proteins. Deletion of CHS5, which encodes a Golgi protein involved in the transport of Chs3p to the cell periphery, is lethal in combination with disruption of SBE2 and SBE22. Thus, we suggest a model in which Sbe2p and Sbe22p are involved in the transport of cell wall components from the Golgi apparatus to the cell surface periphery in a pathway independent of Chs5p.