The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10␣, and ABC10␤), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosagedependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.Eukaryotic transcription is mediated by large multiprotein complexes in which each of the three nuclear RNA polymerases (pols) interact with their cognate preinitiation factors. The pols themselves have been well characterized in terms of subunit composition, especially in the case of the yeast Saccharomyces cerevisiae. However, the spatial organization of the enzyme subunits and the way they interact with preinitiation complexes or with other components of the yeast nucleus are still poorly understood. Electron microscopy so far has provided the most accurate structural description of the Escherichia coli enzyme (1) and of yeast pol I (2, 3) and II (refs. 4-6 and references therein), revealing a striking similarity in the overall shape of these enzymes. In the case of yeast pol I, six subunits (or domains thereof) were localized by immunoelectron microscopy of antibody-labeled enzymes (2, 7). Sitespecific protein-DNA crosslinking also shed light on the general architecture of pol II (8, 9) and III (10-12) transcription complexes.These studies are still far from providing a comprehensive picture of the structural organization of the eukaryotic pols. Alternatively, each subunit can be tested for its ability to selectively associate with other subunits of the same heteromultimeric complex. In the case of human pol II, an in vitro test based on glutathione S-transferase pull-down assays has suggested numerous contacts within the pol II complex (13). In Schizosaccharomyces pombe, studies based on Far Western blotting, which were in some cases supported by independent protein-protein crosslinking studies, suggested that the two large pol II subunits interact with all of the other smaller subunits (9, 14). The two-hybrid system is an alternative to biochemical methods that allows one to detect interactions between proteins in the cellular context of the yeast nucleus (ref. 15 and ...

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.

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 coactivator, respectively. Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts. spt8D and dst1D 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. rpb9D, spt8D and dst1D were lethal in cells lacking the Rpb4 subunit. The TFIIS N-terminal domain is also strictly required for viability in rpb4D, 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.

Summary Rpa12p is a subunit of RNA polymerase I formed of two zinc‐binding domains. The N‐terminal zinc region (positions 1–60) is poorly conserved from yeast to man. The C‐terminal domain contains an invariant Q.RSADE..T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N‐terminal domain fail to grow at 34°C, are sensitive to nucleotide‐depleting drugs and become lethal in rpa14‐Δ mutants lacking the non‐essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two‐hybrid interaction with the second largest subunit. In all these respects, N‐terminal deletions behave like full deletions. In contrast, C‐terminal deletions retaining only the first N‐terminal 60 amino acids are indistinguishable from wild type. Thus, the N‐terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.