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Brf is the TFIIB-related component of Saccharomyces cerevisiae RNA polymerase III transcription initiation factor IIIB (TFIIIB). An extensive set of Brf fragments has been examined for the abilities to assemble the TFIIIB-DNA complex and recruit RNA polymerase III to accurately initiate transcription. The principal TFIIIB-assembly function of Brf was found to be contributed by a C-proximal segment spanning amino acids 435 to 545, while the principal transcription-directing function was contributed by a segment of its N-proximal, TFIIB-homologous half. The diverse activities of Brf were also reconstituted from combined fragments. The fragments spanning amino acids 1 to 282 and 284 to 596 were found to assemble into TFIIIB-DNA and TFIIIC-TFIIIB-DNA complexes that were very stable, transcriptionally highly active, and indistinguishable (by in vitro footprinting) from complexes formed with intact Brf. The proximities of the individual halves of split Brf to DNA were extensively mapped by photochemical cross-linking of the TFIIIB-DNA complex. We also identified sites of interaction of Brf fragments with TATA-binding protein (TBP), taking advantage of a recently completed mutational analysis of the TBP surface. The constraints established by these analyses specify a global model of the functional segments of Brf and how they fit into the structure of the TFIIIB-DNA complex.Eukaryotic nuclear and archaeal transcription apparatuses share an essential common feature: the RNA polymerase (pol) is brought to the transcriptional start site by a DNA-bound assembly of transcription initiation proteins. The promotermarking assembly of archaeal RNA polymerases and the core promoter-marking assembly of eukaryotic pol II each consist of two proteins: TATA-binding protein (TBP) and the phylogenetically related transcription factor B (TFB) or TFIIB (for archaeal or pol II transcription systems, respectively) (43).The situation differs only slightly for transcription by pol III, whose promoter-marking assembly, TFIIIB, is composed of three subunits: TBP, Brf (named for its relatedness to TFIIB) (7,11,33), and BЉ (18,24). In Saccharomyces cerevisiae, the three components of TFIIIB are held together by a more stable interaction between Brf and TBP (which together make up the BЈ component of TFIIIB) and by a weaker interaction between TBP-DNA-bound Brf and BЉ (17, 22); a weaker direct BЉ-TBP interaction has also been noted (12, 37). Each of these TFIIIB components is required for all pol III transcription in yeast. Homologs of Brf and BЉ serve as components of the pol III system of humans (42, 44) but are not ubiquitously required. It appears instead that specialized alternative promoter-marking assemblies participate in different classes of human pol III promoters (16,34,42,48).The homology relationship of Brf with TFIIB is confined to the N-terminal half of the much larger Brf; similarities between the C-proximal half of Brf and proteins of the pol II transcription initiation apparatus have not been discerned, at least at the level of...

Transcription factor IIIB (TFIIIB), the central transcription factor of Saccharomyces cerevisiae RNA polymerase III, is composed of TATA-binding protein, the TFIIB-related protein Brf, and B؆. B؆, the last component to enter the TFIIIB-DNA complex, confers extremely tight DNA binding on TFIIIB. Terminally and internally deleted B؆ derivatives were tested for competence to form TFIIIB-DNA complexes by TFIIIC-dependent and -independent pathways on the SUP4 tRNA Tyr and U6 snRNA (SNR6) genes, respectively, and for transcription. Selected TFIIIB-TFIIIC-DNA complexes assembled with truncated B؆ were analyzed by DNase I footprinting, and the surface topography of B؆ in the TFIIIB-DNA complex was also analyzed by hydroxyl radical protein footprinting. These analyses define functional domains of B؆ and also reveal roles in start site selection by RNA polymerase III and in clearing TFIIIC from the transcriptional start. Although absolutely required for transcription, B؆ can be extensively truncated. Core proteins retaining as few as 176 (of 594) amino acids remain competent to transcribe the SNR6 gene in vitro. TFIIIC-dependent assembly on DNA and transcription requires a larger core of B؆: two domains (I and II) that are required for SNR6 transcription on an either-or basis are simultaneously required for TFIIIC-dependent assembly of DNA complexes and transcription. Domains I and II of B؆ are buried upon assembly of the TFIIIB-DNA complex, as determined by protein footprinting. The picture of the TFIIIB-DNA complex that emerges is that B؆ serves as its scaffold and is folded over in the complex so that domains I and II are near one another.The yeast RNA polymerase III (pol III) transcription machinery is the least complex and, at this time, the most highly resolved of the eukaryotic nuclear transcription systems. As such, it comes closest to supporting a comprehensive and deeper analysis of interactions and mechanisms in the initiation of eukaryotic transcription. The components of its transcription factors, transcription factor IIIA (TFIIIA), TFIIIB, and TFIIIC, are now enumerated and known to be encoded by 10 essential genes (2, 9, 12, 16, 20, 24, 33, 36, 37, 40, 42, 44, 45a, 48). Three of these genes encode the three subunits of its central transcription factor TFIIIB: the TATA-binding protein (TBP), which is required for all nuclear transcription (reviewed in reference 17), Brf, a TFIIB-related 596-amino-acid subunit (also called TFIIIB70), and BЉ (also called TFIIIB90), a 594-amino-acid subunit with no discernible homology to other transcription proteins.The central role of TFIIIB is defined by its ability to recruit pol III to the transcriptional start site (21,25,27). TFIIIB binds very tightly to ϳ25 to 30 bp of greatly varying DNA sequence upstream of the transcriptional start site. It can be brought to these initiation-specifying sites by two mechanisms: through the action of the assembly factor TFIIIC (5, 27) and through direct DNA recognition by its TBP subunit (10, 38, 51). TFIIIC-dependent assembly of TFII...

Arcobacter has emerged as an important food-borne zoonotic pathogen, causing sometimes serious infections in humans and animals. Newer species of Arcobacter are being incessantly emerging (presently 25 species have been identified) with novel information on the evolutionary mechanisms and genetic diversity among different Arcobacter species. These have been reported from chickens, domestic animals (cattle, pigs, sheep, horses, dogs), reptiles (lizards, snakes and chelonians), meat (poultry, pork, goat, lamb, beef, rabbit), vegetables and from humans in different countries. Arcobacters are implicated as causative agents of diarrhea, mastitis and abortion in animals, while causing bacteremia, endocarditis, peritonitis, gastroenteritis and diarrhea in humans. Three species including A. butzleri, A. cryaerophilus and A. skirrowii are predominantly associated with clinical conditions. Arcobacters are primarily transmitted through contaminated food and water sources. Identification of Arcobacter by biochemical tests is difficult and isolation remains the gold standard method. Current diagnostic advances have provided various molecular methods for efficient detection and differentiation of the Arcobacters at genus and species level. To overcome the emerging antibiotic resistance problem there is an essential need to explore the potential of novel and alternative therapies. Strengthening of the diagnostic aspects is also suggested as in most cases Arcobacters goes unnoticed and hence the exact epidemiological status remains uncertain. This review updates the current knowledge and many aspects of this important food-borne pathogen, namely etiology, evolution and emergence, genetic diversity, epidemiology, the disease in animals and humans, public health concerns, and advances in its diagnosis, prevention and control.

The yeast RNA polymerase III transcription machinery consists of three transcription initiation factors, TFIIIA, TFIIIB, and TFIIIC (reviewed in references 15, 24, 28, 45, and 47). A fourth component generates quantitatively more efficient transcription in vitro under certain conditions (13, 41), perhaps by facilitating protein refolding or protein-protein association. The functions of TFIIIB are central to this transcription machinery, because TFIIIB directly recruits RNA polymerase III (pol III) to the transcriptional start site and because TFIIIB and RNA pol III alone suffice for transcriptional initiation (26). TFIIIB has three components, TATA-binding protein (TBP), Brf, and BЉ (8,12,22,27,28,36,40,41); TBP and a protein related to yeast Brf are also components of mammalian TFIIIB; it remains to be seen whether a protein analogous to yeast BЉ also exists in higher eukaryotes (11,35,37,42,43). TBP and Brf, together designated BЈ, are readily separable from BЉ during conventional protein purification (25), suggesting that these two components of TFIIIB are somewhat loosely associated (22), although their binding can be detected by immuno-coprecipitation (39) and by affinity chromatography (23).TFIIIB plays a role in transcription by pol III that TFIIB and TFIID (or TFIIB and TBP) together play in pol II transcription. Like TFIIB and TBP (9, 32, 38), TFIIIB occupies upstream sites on pol III genes that specify the locations of transcriptional start sites. TFIIIB is brought to these sites either through the mediation of an assembly factor, TFIIIC, or by autonomous recognition of specific DNA sequence mediated by its TBP.The genes encoding the subunits of yeast TFIIIB have been identified (28,40,41), making the interactions of these components and their roles in initiation of transcription by pol III far more accessible to analysis by biochemical and molecular genetic methods. When Brf is made in Escherichia coli, internal initiation of translation (34) and proteolytic processing generate numerous fragments. Attachment of an affinity tag at the C-terminal end of Brf allows a subset of these products, which constitute a natural N-terminal deletion series, to be purified. The particular interest of such a series lies in the fact that the homology to TFIIB is confined to the N-terminal half of Brf. Two of the N-deletion forms of Brf that are compared in the experiments below progressively remove the N-terminal zinc finger and the first TFIIB-related repeat in the first instance and both TFIIB homologous repeats together with a segment that is somewhat conserved among fungal Brf (Brf homology I) in the second case. A third deletion variant lacks all of the above and 35 additional amino acids.We have compared these deletion proteins with intact Brf for the ability to interact with the other components of TFIIIB, for the ability to participate in effective recruitment of pol III to a U6 gene promoter, and for ability to be recruited to a tRNA gene by TFIIIC. Our findings suggest a functional complementarity between Br...

TFC5, the unique and essential gene encoding the B" component of the Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been cloned. It (8): the TATA box-binding protein, TBP; the TBP-interacting 67-kDa TFIIBrelated protein, Brf (gene BRFI/PCF4/TDS4) (9-11); and a chromatographically separable component named B" (12). Active B" was previously isolated as a 90-kDa "band" out of denaturing (SDS) gels, renatured, and shown to reconstitute active TFIIIB. In the experiments that are described below, we complete the proof of constitution of TFIIIB by cloning the gene encoding the B" protein, expressing it in Escherichia coli, and using the resulting protein to reconstitute transcriptionally active TFIIIB entirely from its three recombinant constituents.

Transcription factor (TF) IIIB, which directs RNA polymerase (pol) III to its promoters, is made up of three components: the TATA box-binding protein, the TFIIB-related Brf, and the pol III-specific B . Certain mutations in Saccharomyces cerevisiae Brf and B retain TFIIIB transcription factor activity with supercoiled DNA but are inactive with linear duplex DNA. Further analysis shows that these inactive TFIIIB-DNA complexes bind pol III and position it appropriately over the transcriptional start site but do not form DNA strand-separated open promoter complexes. It is proposed that the normal function of TFIIIB combines pol III recruitment with an active role in a subsequent step of transcriptional initiation leading to promoter opening.Yeast RNA polymerase (pol) III is brought to its promoters by its central transcription factor (TF) IIIB, which is composed of three subunits: Brf, its TFIIB-related and archaeal TFBrelated component; TATA box-binding protein (TBP), the ubiquitous component of all eukaryotic nuclear transcription; and BЈЈ, a pol III-specific subunit. All three subunits are required for all transcription by yeast pol III. TFIIIB can bind autonomously to certain pol III promoters through a direct interaction of TBP with a strong TATA box. When such a TATA box is lacking, or when DNA is packaged into chromatin, TFIIIB is brought to the promoter by TFIIIC, its complex, bulky DNA-binding assembly factor (1-9). Once pol III has been recruited to the promoter by TFIIIB, it spontaneously and thermoreversibly generates extensive DNA strand separation (the transcription bubble) around the transcription start site in linear as well as in negatively supercoiled DNA (10).Although it is clearly established that TFIIIB plays the central role in bringing RNA polymerase III to the immediate vicinity of the promoter, the possibility that it also can intervene in subsequent steps of transcriptional initiation has not been explored. The experiments that are described below provide evidence that this is indeed what happens. We show that, when TFIIIB is assembled with certain mutant Brf or BЈЈ subunits, it retains activity for directing transcription of negatively supercoiled DNA or of linear DNA that has been made especially flexible at the TATA box but is inactive for transcription of normal linear duplex DNA. Further analysis shows that pol III is recruited to the inactive TFIIIB-linear DNA complex and is brought into contact with DNA in the vicinity of the transcriptional start site but fails to form a transcription bubble and consequently also fails to make complete or abortive transcripts.We suggest that the complete TFIIIB-DNA complex participates in transcriptional initiation by guiding already recruited pol III through subsequent steps of promoter opening and that certain deficient TFIIIB-DNA complexes fix the already recruited and promoter-proximal polymerase in a conformation that is unable to proceed along the pathway to promoter opening. The observation that pol III can be bound to a promoter in ...

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