Vincent Van Mullem

Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning

The Gateway™ technology is becoming an increasingly popular method for cloning ORFs by recombination. It allows the transfer of any ORF flanked by specific recombination sites into any vectors harbouring the corresponding sites. Here we describe the construction of a set of 20 Saccharomyces cerevisiae Gateway™ compatible vectors. These plasmids bear an URA3 or TRP1 selection marker. They are designed for expression without tag sequence or for C‐ or N‐terminal protein tagging with 3HA (haemagglutinin), 13MYC, 4TAP (tandem affinity purification) or GST (glutathione S‐transferase) epitopes. The centromeric vectors allow expression of DNA sequence in yeast under tetracycline‐regulatable promoters, while expression from the high copy vectors is driven by PGK promoter. To test their applicability, the genes encoding the RNA polymerase I subunit Rpa12p or the TFIIS transcription factor were cloned in these vectors. Their expression was demonstrated using Western blotting or complementation assays. Copyright

Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS.

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two‐hybrid partners of the conserved TFIIS N‐terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co‐activator, respectively. Co‐immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell‐free extracts. spt8Δ and dst1Δ mutants were sensitive to nucleotide‐depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Δ, spt8Δ and dst1Δ were lethal in cells lacking the Rpb4 subunit. The TFIIS N‐terminal domain is also strictly required for viability in rpb4Δ, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8‐containing form of SAGA co‐operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a β-addition motif with the “jaw” of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable fromrpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62–164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-Δ, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.

The asymmetric distribution of the essential histidine kinase PdhS indicates a differentiation event in Brucella abortus

Many organisms use polar localization of signalling proteins to control developmental events in response to completion of asymmetric cell division. Asymmetric division was recently reported for Brucella abortus, a class III facultative intracellular pathogen generating two sibling cells of slightly different size. Here we characterize PdhS, a cytoplasmic histidine kinase essential for B. abortus viability and homologous to the asymmetrically distributed PleC and DivJ histidine kinases from Caulobacter crescentus. PdhS is localized at the old pole of the large cell, and after division and growth, the small cell acquires PdhS at its old pole. PdhS may therefore be considered as a differentiation marker as it labels the old pole of the large cell. Moreover, PdhS colocalizes with its paired response regulator DivK. Finally, PdhS is able to localize at one pole in other α‐proteobacteria, suggesting that a polar structure associating PdhS with one pole is conserved in these bacteria. We propose that a differentiation event takes place after the completion of cytokinesis in asymmetrically dividing α‐proteobacteria. Altogether, these data suggest that prokaryotic differentiation may be much more widespread than expected.

Glucose deprivation mediates interaction between CTDK-I and Snf1 in Saccharomyces cerevisiae

Ctk1 is a kinase involved in transcriptional control. We show in the two‐hybrid system that Ctk1 interacts with Snf1, a kinase regulating glucose‐dependent genes. Co‐purification experiments confirmed the two‐hybrid interaction but only when cells were grown at low glucose concentrations. Deletion of Ctk1 or its associated partners, Ctk2 and Ctk3, conferred synthetic lethality with null mutants of Snf1 or Snf1‐associated proteins. Northern blot analysis suggested that Ctk1 and Snf1 act together in vivo to regulate GSY2. These findings support the view that Ctk1 interacts with Snf1 in a functional module involved in the cellular response to glucose limitation.