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T Ruusala

Publications and source records attributed to T Ruusala.

15 recordsLinked to original sources
PubMed1994

Mutations in rik1, clr2, clr3 and clr4 genes asymmetrically derepress the silent mating-type loci in fission yeast.

In Schizosaccharomyces pombe the mating-type information is stored at two transcriptionally silent loci (mat2 and mat3). The region between these sites (K region) is inert for meiotic crossing over. The mating-type genes (M or P) are expressed only when present at a third, active locus (mat1). We have earlier shown that the positional regulation of P genes is based on repression at the silent site, caused by elements in the flanking DNA sequences. In this study we have mutagenized a sterile mat1 deleted strain and selected for cells that are able to conjugate. Recessive mutations of this type should define genes encoding trans-acting factors involved in repression of the silent mating-type loci. Before this work mutations in two genes, clr1 and swi6, had been shown to allow both expression of the silent loci and recombination in the K region. The sensitivity of the present selection is demonstrated by the isolation of new mutations that derepress one or both of the silent loci (M-mating or bi-mating). The frequency of M-mating mutants was almost two orders of magnitude higher than that of bi-mating mutants and in all mutants analyzed mat3-M expression was significantly higher than mat2-P expression. The mutations define three new genes, clr2, clr3 and clr4. In addition we show that the rik1 mutant previously known to allow recombination in the K region also depresses the silent loci.

Alleles↗
PubMed1993

The silent P mating type locus in fission yeast contains two autonomously replicating sequences.

We show that in fission yeast two DNA fragments at the silent P mating type locus provide plasmids with the capability of autonomous replication. Bacterial vectors containing these sequences replicate in a polymeric form in fission yeast very much like plasmids with the commonly used replication sequence ars1, do. There are, however, several differences between the two new ars sequences. The percentage of cells containing the plasmid during selection, the plasmid copy number and the plasmid segregation during mitosis are all dependent on the choice of the ars sequence. A DNA fragment with ars activity from the left side of the silent P cassette represses the expression of the marker gene, ura4+, at least three hundred fold compared to plasmids containing only the other new ars sequence or only ars1. The importance of replication in this promoter independent transcriptional regulation is further substantiated by the fact that the repression is partially released in the presence of ars1 on the same plasmid.

Genes, Fungal↗
PubMed1992

Sliding and intermolecular transfer of the lac repressor: kinetic perturbation of a reaction intermediate by a distant DNA sequence.

The lac repressor associates with its operator at a rate faster than conventional diffusion allows, either because of one-dimensional diffusion of a captured repressor along the DNA (sliding) or because the tetrameric repressor can be rapidly transferred between DNA sites distant from each other in the primary sequence (direct transfer). We report measurements of relative repressor-operator association rates at physiological operator concentrations. We assay for the ability of DNA targets of equal length (approximately 200 base pairs) containing one or six operator segments to compete for repressor; as the sliding range decreases, the hexameric operator fragment should react up to six times faster than the monomeric operator fragment. We find that the advantage enjoyed by the hexameric fragment varies from little to none at low operator concentration (depending on ionic strength) to more than 3-fold at high concentration. We ascribe this behavior to sliding at low operator concentration and to an increasing contribution of bimolecular direct transfer events as concentration increases. The observations require a "semibound" intermediate state of the protein at operator sites. This species can either undergo a relatively slow (tau greater than 1 sec) unimolecular isomerization to the final complex, or the isomerization can occur in a bridged complex with another operator site, accompanied by transfer to the second operator with probability of 0.5. Bridging alters one or more rate constants in the complex.

Base Sequence↗
PubMed1992

Trans-acting factors and properly positioned DNA elements repress mating-type genes in fission yeast.

Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences. The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region. The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression. Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants. These mutants can de-repress the resistance gene at other positions than the one used for their selection. Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette. These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.

Alleles↗
PubMed1991

Repression of a mating type cassette in the fission yeast by four DNA elements.

The fission yeast, Schizosaccharomyces pombe, expresses one of two alternative mating types. They are specified by one of two determinants (M or P) present at the mat1 locus. In addition, silent copies of M and P are present on the same chromosome. In the present work we demonstrate that the difference between the active and the silent stage of the P determinant is controlled by four repressive elements that are located at the silent locus. There are two elements to the left and two to the right of the mating type cassette. Both elements to the left and either one of the two elements to the right are required for an effective blockage of transcription. When they are combined, the four elements define a highly efficient silencer functionally similar to the HMRE and HMLE and HMLI silencers in Saccharomyces cerevisiae. In addition, the DNA surrounding the silent P locus confers symmetric partitioning in mitosis to Schizosaccharomyces pombe ars plasmids.

DNA, Fungal↗
PubMed1991

The mating type in fission yeast is switched independently of its expression.

The mating type of fission yeast is determined by the mat1 locus on chromosome II. The sequence content of this locus, and hence the mating type, is switched in a strictly regular pattern by transposition from one of two unexpressed mating type sequences. The expressed and the two silent sequences are located on the same chromosome. It is not understood how one of the two donor sequences is selected in this reaction. Here I test the possibility that the selection is governed by gene expression from the mat1 locus. Such a mechanism could favor transposition of a donor sequence of opposite mating type to the one present at mat1. Alternatively it could disfavor transposition of a synonymous sequence. The present data argue strongly against any type of participation of mat1 gene products in the choice of donor during the mating type switch. Alternative steering mechanisms are discussed.

Blotting, Northern↗
PubMed1987

Does streptomycin cause an error catastrophe?

We have examined the interpretation that streptomycin kills a bacterial culture by initiating the so-called error catastrophe. In particular, we asked whether the increased translational error rate induced by the antibiotic gives rise to an autocatalytic loss of functional fidelity of the devices responsible for gene expression, which ultimately causes the death of the culture. We have analyzed the performance characteristics of one of these devices, namely the ribosome in streptomycin-treated bacteria. We find that, although the treated ribosomes are constructed from error-containing proteins, they are not significantly different in elongation rate and fidelity from those ribosomes taken from untreated bacteria. We conclude that the bacteriocidal effect of streptomycin is not due to the initiation of an error catastrophe.

Dihydrostreptomycin Sulfate↗
PubMed1986

Counting cycles of EF-Tu to measure proofreading in translation.

A new method (T. Ruusala et al., 1982, EMBO J. 1, 75-78, 741-748) for analyzing kinetic proofreading in translation is described. An in vitro system is arranged so that its rate of polypeptide synthesis is determined by the release rate of GDP from EF-Tu in the absence of EF-Ts. This enables the counting of the number of EF-Tu cycles for correct as well as for incorrect peptide bonds. The necessary equations are derived and the approximations involved in these are discussed together with data from experiments not previously described.

Guanosine Diphosphate↗
PubMed1986

The homeo domain of a murine protein binds 5' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.

Amino Acid Sequence↗
PubMed1984

Hyper-accurate ribosomes inhibit growth.

We have compared both in vivo and in vitro translation by ribosomes from wild-type bacteria with those from streptomycin-resistant (SmR), streptomycin-dependent (SmD) and streptomycin-pseudo-dependent (SmP) mutants. The three mutant bacteria translate more accurately and more slowly in the absence of streptomycin (Sm) than do wild-type bacteria. In particular, the SmP bacteria grow at roughly half the rate of the wild-type in the absence of Sm. The antibiotic stimulates both the growth rate and the translation rate of SmP bacteria by approximately 2-fold, but it simultaneously increases the nonsense suppression rate quite dramatically. Kinetic experiments in vitro show that the greater accuracy and slower translation rates of mutant ribosomes compared with wild-type ribosomes are associated with much more rigorous proofreading activities of SmR, SmD and SmP ribosomes. Sm reduces the proofreading flows of the mutant ribosomes and stimulates their elongation rates. The data suggest that these excessively accurate ribosomes are kinetically less efficient than wild-type ribosomes, and that this inhibits mutant growth rates. The stimulation of the growth of the mutants by Sm results from the enhanced translational efficiency due to the loss of proofreading, which more than offsets the loss of accuracy caused by the antibiotic.

Drug Resistance, Microbial↗
PubMed1984

Kinetic impairment of restrictive streptomycin-resistant ribosomes.

Comparisons in vivo and in vitro of wild-type and otherwise isogenic bacteria with five different mutant alleles of the gene (rpsL) specifying ribosomal protein S12, all resistant to high levels of streptomycin, show that the streptomycin-resistant (Smr) phenotype can be subdivided into major groups: restrictive and non-restrictive. The restrictive bacteria have a characteristically lower frequency of nonsense suppression in vivo, and are also slower than the wild type in their rate of protein synthesis. Non-restrictive Smr bacteria on the other hand do not differ significantly from the wild type either in nonsense suppression frequencies or in the rate of translation. A complementary pattern is seen in vitro, where ribosomes from the restrictive Smr bacteria translate poly(U) with a significantly lower missense error frequency than wild-type ribosomes, and also show an increased Michaelis constant (KM) with respect to their substrate, i.e. ternary complexes. Both effects are correlated with the more aggressive proofreading function that is characteristic of these restrictive ribosomes. In contrast, ribosomes isolated from the non-restrictive Smr bacteria do not show any major difference in either proofreading or missense error in vitro when compared to the wild type.

Drug Resistance, Microbial↗
PubMed1984

Streptomycin preferentially perturbs ribosomal proofreading.

We have studied the influence of streptomycin (Sm) on the kinetics and accuracy of translation by wild-type as well as Ram-mutant ribosomes in an in vitro system that mimics the performance characteristics of ribosomes in bacteria. It can be shown in this system that the accuracy of translation is made up of an initial selection step and one or more proofreading steps. The data show that the antibiotic has only a small influence on the initial selectivity step of wild-type or mutant ribosomes. Streptomycin stimulates the missense rate primarily by suppressing the proofreading of the ribosomes. The kinetic effects of Sm and of Ram alteration are not additive, but seem to be overlapping if not identical.

Escherichia coli↗
PubMed1982

Catalytic effects of elongation factor Ts on polypeptide synthesis.

The kinetic parameters which characterize the interaction between elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) have been determined in a poly(uridylic acid)-primed translation system. The EF-Ts catalyzed release of GDP from EF-Tu was measured independently in a nucleotide exchange assay. We conclude that the rate-limiting step for the EF-Tu cycle in protein synthesis in the absence of EF-Ts is the release of GDP. By adding EF-Ts the time of this step is reduced from 90 s to 30 ms. Half maximal rate is obtained at an EF-Ts concentration of 2.5 x 10 M.

Journal Article↗
PubMed1982

Is there proofreading during polypeptide synthesis?

The stoichiometric efficiency with which ternary complexes containing Phe-tRNAphe and Leu-tRNAleu support polypeptide synthesis has been compared in a poly(U)-directed, steady-state translation system. When unfractionated tRNA is used to support synthesis, the number of discharged ternary complexes per peptide bond formed is an average of 48 times greater for leucine than for phenylalanine. When three purified leucine isoacceptor species are tested, they each show a characteristic ratio of ternary complexes discharged per missense insertion, normalized to that for phenylalanine: these are 103, 76, and 45 for Leu- tRNA2leu , Leu- tRNA3leu , and Leu- tRNA4leu , respectively. The data are consistent with the functioning of a proofreading mechanism during translation.

Escherichia coli↗