Gregor Högenauer

The gene encoding squalene epoxidase from Saccharomyces cerevisiae: cloning and characterization.

The gene (ERG1) encoding squalene epoxidase (ERG) from Saccharomyces cerevisiae was cloned. It was isolated from a gene library, prepared from an allylamine-resistant (AlR) S. cerevisiae mutant, by screening transformants in a sensitive strain for AlR colonies. The ERG tested in a cell-free extract from one of these transformants proved to be resistant to the Al derivative, terbinafine. From this result, we concluded that the recombinant plasmid in the transformant carried an allelic form of the ERG1 gene. The nucleotide sequence showed the presence of one open reading frame coding for a 55,190-Da peptide of 496 amino acids. Southern hybridization experiments allowed us to localize the ERG1 gene on yeast chromosome 15.

Diazaborine resistance in the yeast Saccharomyces cerevisiae reveals a link between YAP1 and the pleiotropic drug resistance genes PDR1 and PDR3

We have investigated the mechanisms underlying resistance to the drug diazaborine in Saccharomyces cerevisiae. We used UV mutagenesis to generate resistant mutants, which were divided into three different complementation groups. The resistant phenotype in these groups was found to be caused by allelic forms of the genes AFG2, PDR1, andPDR3. The AFG2 gene encodes an AAA (ATPases associated to a variety of cellularactivities) protein of unknown function, whilePDR1 and PDR3 encode two transcriptional regulatory proteins involved in pleiotropic drug resistance development. The isolated PDR1–12 and PDR3–33alleles carry mutations that lead to a L1044Q and a Y276H exchange, respectively. In addition, we report that overexpression of Yap1p, the yeast homologue of the transcription factor AP1, results in a diazaborine-resistant phenotype. The YAP1-mediated diazaborine resistance is dependent on the presence of functionalPDR1 and PDR3 genes, although PDR3had a more pronounced effect. These results provide the first evidence for a functional link between the Yap1p-dependent stress response pathway and Pdr1p/Pdr3p-dependent development of pleiotropic drug resistance.

Cytoplasmic recycling of 60S preribosomal factors depends on the AAA protein Drg1.

ABSTRACT Allelic forms of DRG1/AFG2 confer resistance to the drug diazaborine, an inhibitor of ribosome biogenesis in Saccharomyces cerevisiae. Our results show that the AAA-ATPase Drg1 is essential for 60S maturation and associates with 60S precursor particles in the cytoplasm. Functional inactivation of Drg1 leads to an increased cytoplasmic localization of shuttling pre-60S maturation factors like Rlp24, Arx1, and Tif6. Surprisingly, Nog1, a nuclear pre-60S factor, was also relocalized to the cytoplasm under these conditions, suggesting that it is a previously unsuspected shuttling preribosomal factor that is exported with the precursor particles and very rapidly reimported. Proteins that became cytoplasmic under drg1 mutant conditions were blocked on pre-60S particles at a step that precedes the association of Rei1, a later-acting preribosomal factor. A similar cytoplasmic accumulation of Nog1 and Rlp24 in pre-60S-bound form could be seen after overexpression of a dominant-negative Drg1 variant mutated in the D2 ATPase domain. We conclude that the ATPase activity of Drg1 is required for the release of shuttling proteins from the pre-60S particles shortly after their nuclear export. This early cytoplasmic release reaction defines a novel step in eukaryotic ribosome maturation.

TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network

Site‐directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1‐mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1‐16 plasmid, a transfer‐derepressed (finO −) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1‐16 was 107‐fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co‐ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.

Repression and derepression of conjugation of plasmid R1 by wild‐type and mutated finP antisense RNA

The finP gene of plasmid R1 is located between the genestraM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5′ non‐translated sequences, the ribosome‐binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1‐19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R l‐19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1‐19 intrans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.

The FinOP repressor system of plasmid R1: analysis of the antisense RNA control of traJ expression and conjugative DNA transfer

A key determinant of the frequency of IncF plasmid‐mediated DNA transfer between enterobacterial cells is the FinOP system. traJ, a positive regulator of the transfer (tra ) genes is controlled at the post‐transcriptional level by two negative elements, finP and finO. FinP is a plasmid‐specific antisense RNA, whereas finO encodes a proteic co‐repressor which is not plasmid specific but exchangeable among F‐like plasmids. We designed a traJ–lacZ test system that allowed us to monitor the effects of FinP and various FinP mutants on traJ expression. Furthermore, the introduction of finO into the test system enabled us to assess the function of FinO in the interaction of FinP with its target, the traJ mRNA. In this test system, FinP, expressed from a single‐copy plasmid, in the absence of FinO, repressed traJ expression six‐fold. When expressed from a pBR322‐derived multicopy plasmid FinP repressed traJ expression approx. 2000‐fold. This result unambiguously demonstrated that FinP is sufficient to repress traJ expression in a gene dosage‐dependent manner. Mutations of finP creating base exchanges either in loop I or loop II of the two stem‐loop structures of the antisense RNA led to a dramatic decrease in the repressor activity. In a combined loop I–loop II mutation the repressor activity was almost completely lost, supporting the model that the first critical interaction between the two RNA molecules occurs via ‘kissing’ of both loops of the RNAs. Addition of finO to the test system enhanced the repression of traJ expression by FinP by up to two orders of magnitude. This effect of FinO on FinP activity in vivo might indicate that FinO, in addition to its function as an RNA stabilizer, promotes complex formation between the target mRNA and the antisense RNA. Such a function of FinO has recently been shown to exist in vitro (van Biesen and Frost (1994) Mol Microbiol14: 427–436).

The TraM protein of plasmid R1 is a DNA‐binding protein

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA‐binding studies. Both gel retardation‐and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.

TraM of plasmid R1 regulates its own expression

Regulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusions. tram″lacZ fusion constructs were transferred to the chromosome via the recombinant phage λRZ5. The level of β‐galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five‐ to sixfold when the single‐copy plasmids R1 or R1‐19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45‐fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatjvely repressed or derepressed. Site‐specific mutagenesis of the region encoding the N‐terminus of the TraM protein eliminated the autoregulative effect indicating that the N‐terminal amino acids of the protein are important to its DNA‐binding function. The autoregulatory behaviour of TraM is allele specific. R1‐ or P307‐encoded TraM molecules were found to recognize only the cognate DNA.

The kdsA gene coding for 3-deoxy-D-manno-octulosonic acid 8-phosphate synthetase is part of an operon in Escherichia coli

SummaryThe kdsA gene of Escherichia coli was sequenced. It consists of 284 codons and is the last gene of a larger transcription unit. The mRNA is terminated by a rho-independent termination signal with the potential to be active in both orientations. This region is followed by another termination signal which seems to be active in the opposite orientation. Upstream of the kdsA gene a second open reading frame (ORF) was found; both this and the kdsA gene are transcribed in the same mRNA molecule when coded from the chromosome. In a plasmid-carried insert transcription starts from a cryptic promoter within the ORF preceding the kdsA gene. This promoter is not active in the intact chromosome.

Expression of gene 19 of the conjugative plasmid R1 is controlled by RNase III

Specific cleavage of mRNAs by RNase III has been shown to control the expression of several Escherichia coli genes. We show here that the expression of gene 19 of the conjugative resistance plasmid R1 is controlled in its expression by the same endoribonuclease. In vivo studies revealed that a DNA fragment of 150 nucleotides including a perfect 22 nucleotide inverted repeat in the gene 19 coding region is responsible for the low expression of the gene both at the protein and the RNA levels. By using a translational gene 19‐lacZ fusion in isogenic RNase III+ and RNase III‐ strains we could identify RNase III as the key element in the down‐regulation of gene 19 expression. The sequencing of in vitro generated and RNase Ill‐digested transcripts confirmed the in vivo studies and revealed the exact positions of the RNase III cleavage sites within the coding part of the gene 19 transcript. The in vitro determined RNase III cleavage of gene 19 mRNA was confirmed by in vivo primer extension analysis. Finally, we could show that an exchange of three nucleotides within the RNase III recognition site abolished RNase III cleavage in vitro.